Tandem Repeat Regions Of The Human Mucin Muc In Pancreatic Cancer – Monoclonal Antibodies Recognizing The Non: I Know It’s Also A Potential Biomarker For The Diagnosis

MUC4 is expressed in various epithelial tissues, including the epithelia of fetal lungs and the adult respiratory tract from the trachea to the collecting ducts lung trachea, colon, endocervix, conjunctiva, cornea, salivary glands, middle ear and eustachian tube.

These studies from our laboratory and similar groups indicate the potential importance of this mucin in various facts of tumor biology. Whenever indicating its role in disease development, In recent studies, a progressive increase in MUC4 expression had been observed in pancreatic intraepithelial neoplastic lesions. Further, our recent studies have demonstrated that MUC4 results in oncogenic transformation of mouse fibroblasts, contributes to the drugresistance of pancreatic cancer cells by activating anti apoptotic pathways, and is involved in the epithelialtomesenchymal transition in ovarian cancer cells. Now look. Previous studies from our laboratory have shown that inhibition of MUC4 expression using ‘antisense’ or shortinterfering RNA oligonucleotides specific to MUC4 results in a decreased tumorigenicity and dissemination of cancer cells.

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Protein lysates from the MUC4expressing HPAF/CD18 cells were immunoprecipitated using 5 µg/ml of 2382, 2214, 2175, 8G7, and K2G6.

For MUC4 immunodetection, anti MUC4″ mouse monoclonal antibody 8G7 positive control, and 2 µg/ml of non tandem repeat antibodies diluted in PBS were used. It is western blotting using standard procedures. The samples were resolved on 2percentage SDSagarose gel and were immunoblotted using 8G7. Whenever containing protease inhibitor mixture and phosphatase inhibitors, and kept at 4°C for at least 30 min, the cells were washed twice in PBS and scraped in radioimmunoprecipitation assay buffer. Resolved proteins were transferred onto the polyvinylidene difluoride membrane and subjected to the standard immunodetection procedure using specific antibodies. Basically, anti human βactin secondary antibody was used at a dilution of 1∶The blots were processed with ECL Chemiluminescence kit, and the signal was detected by exposing the processed blots to Xray films. SDSPAGE’ was used for ‘β actin’,, and run under similar conditions. Antigen Antibody complexes formed were pulled down by using Protein A/G beads and the complexes were solublized by using ‘SDSsample’ buffer containing ‘2 mercaptoethanol’. Cell lysates were passed through the needle syringe or alternatively subjected to one freezethaw cycle to facilitate the disruption of the cell membranes. As a result, cell lysates were centrifuged at 14000 rpm for 30 min at 4°C, and supernatants were collected. Although, the proteins were resolved by electrophoresis on a 2 SDS agarose gel under reducing conditions, as long as of the large size of MUC4. Seriously. Protein concentrations were determined using a ‘BIO RAD’ D/C protein estimation kit.

For flow cytometry, cells were harvested ‘nonenzymatically’ using Cellstripper, washed with PBS and counted. Slides were washed with PBST and incubated with the ABC solution. All slides were observed under Nikon E400 Light Microscope and representative photographs were taken. The slides were washed with water and counterstained with hematoxylin. Notice, sections were hereafter incubated with the anti MUC4 antibody followed by incubation with secondary antibody for 30 min. Sections were cut and processed as described previously. Briefly, tissue sections were deparaffinized in xylene, and rehydrated in graded ethanol. It’s a well the sections were after that, dehydrated in graded alcohols and mounted with Permount permanent mounting media. The reaction color was developed by incubating sections with 33′-diaminobenzidine reagent. Subsequently, cells were washed three times with PBS and incubated with FITC conjugated ‘antimouse’ antibody for 1 h on ice. Cells were consequently incubated with indicated antibodies for 1 h on ice. Cells were fixed for 30 min with 2 paraformaldehyde and blocked with 5percent goat serum. Cells were washed again three times with PBS and analyzed using the BD FACSCalibur flow cytometer. Usually, nonspecific binding was blocked by incubating the sections with normal ‘goat serum’ for 30 min at room temperature. Furthermore, endogenous peroxidase activity was quenched by incubating sections in 3 H2O2 in PBS for 20 min. Tissues were fixed in 10percent buffered formalin and embedded in paraffin. Schematic structure of MUC4 and recombinant proteins used in the study. For cell surface staining, parformaldehydefixed cells were used and the binding of the antibodies was analyzed by flow cytometry. No staining was observed with Mab 2106 or the nonspecific isotype matched control MAb K2G6. Fractions containing pure GST fusion proteins were pooled and quantified using the BIORAD D/C protein estimation kit. YTA medium, and grown under agitation at 37C for 3 to 4 h to reach an absorbance at 260 nm between 6 8″, induced by 1 IPTG mM, and cultured for an addition of 3 to 4 Cultures were centrifuged and washed three times in ice cold PBS, resuspended in 5 ml of ice cold PBS, and sonicated. None of the tested antibodies except 2214, immunopecipitated the MAb 2214 reactive low molecular weight type of MUC4. Figure 5). ‘TM transmembrane’ domain; CT cytoplasmic tail, Cys cystein rich domain ‘EGF epidermal’ growth factor like domain. Figure 6). ELISA showing the reactivity of ‘anti MUC4’ MAbs to recombinant immunogens. The assay also included a ‘non specific’ isotype matched control K2G6. While MAb 2382 also resulted in considerable enrichment of the 8G7 reactive protein bands, the immunoprecipitated samples from various antibodies were also immunoblotted with MAb 2214 due to its predominant reactivity with a lower molecular weight sort of MUCWhen probed with MAb 8G7, the highest quantity of MUC4 was immunoprecipitated with 8G7. The constructs were sequenced to confirm the proper reading frame and maintained in coli BL21. Whenever allowing ‘in frame’ cloning with the GST and thrombin cleavage site of the ‘pGEX 2TK’ vector, BamHI and a EcoRI restriction sites were added in the forward and reverse primers. Choudhury et al in accordance with the original numbering. Elution fractions of 1 ml were collected and 5 µl aliquot of any fraction was resolved on 10percentage SDSPAGE, and proteins detected by coomassie blueish staining. Further, none of the antibodies showed any reactivity with MUC4 negative pancreatic cancer cell lines MiaPaCa or Panc1. Amplification was done by the expand long RT PCR system as described previously using JER103 and JER109 as templates for sequence AJ00281 and AJ010901. Figure 3). Figure 1a). While the surface reactivity of MAbs 2175 and 2382 was weak and the mean fluorescence intensity values were comparable to the values obtained with MAb 8G7, mAb 2214 exhibited the strongest reactivity with the cell surface in paraformaldehydefixed cells. Protein lysates were clarified by centrifugation and by filtration on a 22 µm filter. Recombinant domains of MUC4α corresponding to the fragments upstream and downstream of the tandem repeat domain were cloned and expressed as described in Materials and Methods and termed MUC4αNter and MUC4 α C Ter. The indicated MAbs were incubated with the 5 µg/ml of GST tagged ‘N terminal’ and tandem repeat recombinant domains of MUCThe specificities were also tested against the MUC4 TR peptide, GST and a non specific control protein bovine serum albumin and the antibodies exhibited negative reactivity against these antigens. MUC4 is putatively cleaved at the GDPH site to generate an N terminal mucin type subunit MUC4α and a Cterminal growth ‘factortype’ subunit MUC4Important domains of MUC4 are marked. Author Contributions. MAbs 2175 and 2214 also immunoprecipitated the ‘full length’ 8G7 reactive band but the enrichment was not as strong as observed with MAbs 8G7 and AntiCterminal MAb 2106 and negative control anti KLH antibody K2G6 did not pull down any 8G7 reactive protein band. While MAbs 2175, mAb 2214 showed a both membrane and perinuclear staining, 2382 and 2106 showed cytoplasmic and membrane staining. The pattern of staining with the new antibodies was similar to that observed with 8G7 showing diffuse staining in both the membrane and the cytoplasm of the tumor cells. Lysates were passed through a 5 ml Glutathione Sepharose Fast Flow column, washed three times with 5 column volumes of PBS, and eluted with 10 15 ml mM reduced gluthatione. The nucleotide numbers corresponding to the boundaries of the recombinant domains are marked and are described in Moniaux et al. The antiTR MAb 8G7 showed strongest reactivity since the repetitive nature of the epitopes. Schematic structure of MUC4 and recombinant proteins used in the study. For cell surface staining, parformaldehydefixed cells were used and the binding of the antibodies was analyzed by flow cytometry. No staining was observed with Mab 2106 or the nonspecific isotype matched control MAb K2G6. Fractions containing pure GST fusion proteins were pooled and quantified using the BIORAD D/C protein estimation kit. YTA medium, and grown under agitation at 37C for 3 to 4 h to reach an absorbance at 260 nm between 6 8″, induced by 1 IPTG mM, and cultured for an addition of 3 to 4 Cultures were centrifuged and washed three times in ice cold PBS, resuspended in 5 ml of ice cold PBS, and sonicated. None of the tested antibodies except 2214, immunopecipitated the MAb 2214 reactive low molecular weight kind of MUC4. Figure 5). ‘TM transmembrane’ domain; CT cytoplasmic tail, Cys cystein rich domain ‘EGF epidermal’ growth factor like domain. Figure 6). ELISA showing the reactivity of ‘anti MUC4’ MAbs to recombinant immunogens. The assay also included a ‘non specific’ isotype matched control K2G6. While MAb 2382 also resulted in considerable enrichment of the 8G7 reactive protein bands, the immunoprecipitated samples from various antibodies were also immunoblotted with MAb 2214 due to its predominant reactivity with a lower molecular weight type of MUCWhen probed with MAb 8G7, the highest percentage of MUC4 was immunoprecipitated with 8G7. The constructs were sequenced to confirm the proper reading frame and maintained in coli BL21. While allowing ‘in frame’ cloning with the GST and thrombin cleavage site of the ‘pGEX 2TK’ vector, BamHI and a EcoRI restriction sites were added in the forward and reverse primers. Choudhury et al in accordance with the original numbering. Elution fractions of 1 ml were collected and 5 µl aliquot of any fraction was resolved on 10 SDSPAGE, and proteins detected by coomassie blueish staining. Further, none of the antibodies showed any reactivity with MUC4 negative pancreatic cancer cell lines MiaPaCa or Panc1. Amplification was done by the expand long RT PCR system as described previously using JER103 and JER109 as templates for sequence AJ00281 and AJ010901. Figure 3). Figure 1a). While the surface reactivity of MAbs 2175 and 2382 was weak and the mean fluorescence intensity values were comparable to the values obtained with MAb 8G7, mAb 2214 exhibited the strongest reactivity with the cell surface in paraformaldehydefixed cells. Protein lysates were clarified by centrifugation and by filtration on a 22 µm filter. Recombinant domains of MUC4α corresponding to the fragments upstream and downstream of the tandem repeat domain were cloned and expressed as described in Materials and Methods and termed MUC4αNter and MUC4 α C Ter. The indicated MAbs were incubated with the 5 µg/ml of GST tagged ‘N terminal’ and tandem repeat recombinant domains of MUCThe specificities were also tested against the MUC4 TR peptide, GST and a non specific control protein bovine serum albumin and the antibodies exhibited negative reactivity against these antigens. MUC4 is putatively cleaved at the GDPH site to generate an N terminal mucin type subunit MUC4α and a Cterminal growth ‘factortype’ subunit MUC4Important domains of MUC4 are marked. Author Contributions. MAbs 2175 and 2214 also immunoprecipitated the ‘full length’ 8G7 reactive band but the enrichment was not as strong as observed with MAbs 8G7 and AntiCterminal MAb 2106 and negative control anti KLH antibody K2G6 did not pull down any 8G7 reactive protein band. While MAbs 2175, mAb 2214 showed a both membrane and perinuclear staining, 2382 and 2106 showed cytoplasmic and membrane staining. The pattern of staining with the new antibodies was similar to that observed with 8G7 showing diffuse staining in both the membrane and the cytoplasm of the tumor cells. Lysates were passed through a 5 ml Glutathione Sepharose Fast Flow column, washed three times with 5 column volumes of PBS, and eluted with 10 15 ml mM reduced gluthatione. The nucleotide numbers corresponding to the boundaries of the recombinant domains are marked and are described in Moniaux et al. The antiTR MAb 8G7 showed strongest reactivity since the repetitive nature of the epitopes.

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