MA5-11202

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of MUC-1 (CT2) was performed by loading 25 µg of HeLa (left lane) and MCF7 (right lane) cell lysates, and 10 µL of PageRuler Plus Prestained Protein Ladder (Product # 26619) onto a Novex® 4-20% Tris-Glycine polyacrylamide gel. Proteins were transferred to a PVDF membrane using the G2 Fast Blotter (Product # 62288), and blocked with 5% milk in TBST for 1 hour at room temperature. Small subunits of MUC-1 were detected between ~14 kD to 30 kD mainly in MCF7 breast cancer cell samples using a MUC-1 monoclonal antibody (Product # MA5-11202) at a concentration of 1 µg/mL overnight at 4C on a rocking platform, followed by a goat anti-Armenian hamster IgG-HRP secondary antibody (Product # PA1-32045) at a dilution of 1:10,000 for at least 1 hour at room temperature. Chemiluminescent detection was performed using SuperSignal West Dura (Product # 34076).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of MUC-1 (CT2) was performed by loading 25 µg of T47D cell lysates, and 10 µL of PageRuler Plus Prestained Protein Ladder (Product # 26619) onto a Novex® 4-20% Tris-Glycine polyacrylamide gel. Proteins were transferred to a PVDF membrane using the G2 Fast Blotter (Product # 62288), and blocked with 5% milk in TBST for 1 hour at room temperature. Small subunits of MUC-1 were detected between ~14 kD to 30 kD using a MUC-1 monoclonal antibody (Product # MA5-11202) at a concentration of 1 µg/mL overnight at 4C on a rocking platform, followed by a goat anti-Armenian hamster IgG-HRP secondary antibody (Product # PA1-32045) at a dilution of 1:10,000 for at least 1 hour at room temperature. Chemiluminescent detection was performed using SuperSignal West Dura (Product # 34076).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using Anti-MUC1 Monoclonal Antibody (MH1 (CT2)), (Product # MA5-11202) and ~19 kDa band corresponding to MUC1 was observed across cell lines tested except in MCF7, HCT 116 and IMR-32 which are reported to be negative. Whole cell extracts (30 µg lysate) of T47D (Lane 1), MCF7 (Lane 2), DU145 (Lane 3), HCT 116 (Lane 4) and IMR-32 (Lane 5) were electrophoresed using Novex® NuPAGE® 4-12 % Bis-Tris gel (Product # NP0321BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (1µg/mL) and detected by chemiluminescence with Rabbit anti-Hamster IgG (H+L) Secondary Antibody, HRP (Product # A18889, 1:4000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Super Signal™ West Dura Extended Duration Substrate (Product # 34076).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescent analysis of CD227/MUC1/Mucin 1 (green) showing staining in the in the cytoplasm and membrane of NIH-3T3 cells (right) compared to a negative control without primary antibody (left). Formalin-fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 5-10 minutes and blocked with 3% BSA-PBS for 30 minutes at room temperature. Cells were probed with a CD227/MUC1/Mucin 1 monoclonal antibody (Product # MA5-11202) in 3% BSA-PBS at a dilution of 1:50 and incubated overnight at 4ºC in a humidified chamber. Cells were washed with PBST and incubated with a DyLight-conjugated secondary antibody in PBS at room temperature in the dark. F-actin (red) was stained with a fluorescent red phalloidin and nuclei (blue) were stained with Hoechst or DAPI. Images were taken at a magnification of 60x.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescent analysis of CD227/MUC1/Mucin 1 (green) showing staining in the in the cytoplasm and membrane of MCF-7 cells (right) compared to a negative control without primary antibody (left). Formalin-fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 5-10 minutes and blocked with 3% BSA-PBS for 30 minutes at room temperature. Cells were probed with a CD227/MUC1/Mucin 1 monoclonal antibody (Product # MA5-11202) in 3% BSA-PBS at a dilution of 1:50 and incubated overnight at 4ºC in a humidified chamber. Cells were washed with PBST and incubated with a DyLight-conjugated secondary antibody in PBS at room temperature in the dark. F-actin (red) was stained with a fluorescent red phalloidin and nuclei (blue) were stained with Hoechst or DAPI. Images were taken at a magnification of 60x.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of CD227/MUC1/Mucin 1 (green) showing staining in the in the cytoplasm and membrane of NIH-3T3 cells (right) compared to a negative control without primary antibody (left). Formalin-fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 5-10 minutes and blocked with 3% BSA-PBS for 30 minutes at room temperature. Cells were probed with a CD227/MUC1/Mucin 1 monoclonal antibody (Product # MA5-11202) in 3% BSA-PBS at a dilution of 1:50 and incubated overnight at 4ºC in a humidified chamber. Cells were washed with PBST and incubated with a DyLight-conjugated secondary antibody in PBS at room temperature in the dark. F-actin (red) was stained with a fluorescent red phalloidin and nuclei (blue) were stained with Hoechst or DAPI. Images were taken at a magnification of 60x.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of CD227/MUC1/Mucin 1 (green) showing staining in the in the cytoplasm of T47D cells (right) compared to a negative control without primary antibody (left). Formalin-fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 5-10 minutes and blocked with 3% BSA-PBS for 30 minutes at room temperature. Cells were probed with a CD227/MUC1/Mucin 1 monoclonal antibody (Product # MA5-11202) in 3% BSA-PBS at a dilution of 1:50 and incubated overnight at 4ºC in a humidified chamber. Cells were washed with PBST and incubated with a DyLight-conjugated secondary antibody in PBS at room temperature in the dark. F-actin (red) was stained with a fluorescent red phalloidin and nuclei (blue) were stained with Hoechst or DAPI. Images were taken at a magnification of 60x.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescence analysis of MUC1 (green) in lactating mouse mammary tissue. FFPE sections were subjected to antigen retrieval at pH 6.0 followed by blocking with 1% BSA and 10% goat serum. Cells were stained with monoclonal MUC1 antibody (Product # MA5-11202) at a dilution of 1:50 at 4 deg overnight, followed by Armenian hamster 488 secondary antibody (green) and DAPI (blue) to stain the nuclei. Image courtesy of Antibody Data Exchange Program.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Flow cytometry analysis of Mucin 1 in NIH-3T3 cells (green) compared to an isotype control (blue). Cells were harvested, adjusted to a concentration of 1-5x10^6 cells/mL, fixed with 2% paraformaldehyde and washed with PBS. Cells were blocked with a 2% solution of BSA-PBS for 30 min at room temperature and incubated with a Mucin 1 monoclonal antibody (Product # MA5-11202) at a dilution of 2 µg/test for 40 min at room temperature. Cells were then incubated for 40 min at room temperature in the dark using a Dylight 488-conjugated secondary antibody and re-suspended in PBS for FACS analysis.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Flow cytometry analysis of Mucin 1 in T47D cells (green) compared to an isotype control (blue). Cells were harvested, adjusted to a concentration of 1-5x10^6 cells/mL, fixed with 2% paraformaldehyde and washed with PBS. Cells were blocked with a 2% solution of BSA-PBS for 30 min at room temperature and incubated with a Mucin 1 monoclonal antibody (Product # MA5-11202) at a dilution of 1 µg/test for 40 min at room temperature. Cells were then incubated for 40 min at room temperature in the dark using a Dylight 488-conjugated secondary antibody and re-suspended in PBS for FACS analysis.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Flow cytometry analysis of Mucin 1 in MCF-7 cells (green) compared to an isotype control (blue). Cells were harvested, adjusted to a concentration of 1-5x10^6 cells/mL, fixed with 2% paraformaldehyde and washed with PBS. Cells were blocked with a 2% solution of BSA-PBS for 30 min at room temperature and incubated with a Mucin 1 monoclonal antibody (Product # MA5-11202) at a dilution of 2 µg/test for 40 min at room temperature. Cells were then incubated for 40 min at room temperature in the dark using a Dylight 488-conjugated secondary antibody and re-suspended in PBS for FACS analysis.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig 3 A) Body weight and kidney weight as percent of body weight (%KW/BW) in wild-type mice and in Pde1a Del15 and Pde1a InsA homozygous mice or data from both combined. B) Axial and coronal MR images of Pde1a mutant mice at 6 and 12 months of age showing small renal cysts (arrows; this panel is also shown as a larger figure in Fig B in S1 File ). C) Kidney sections from Pde1a Del15 and Pde1a InsA homozygous mice showing multiple small cysts staining positively for Tamm-Horsfall protein and epithelial membrane antigen, less consistently for aquaporin-2, and not staining for lysozyme. D) Reduced urine osmolality after 24 hours of dehydration, faster excretion of an acute water load, and E) reduced expression of glycosylated (upper band) and unglycosylated (lower band) pSer269-AQP2 in Pde1a Del15 and Pde1a InsA homozygotes compared to wild-type mice. One-way ANOVA with post-hoc Tukey test was used for the statistical analysis.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig 5 A) Upper panels: Kidney weights as percent of body weights (%KW/BW) and renal cAMP levels in untreated or desmopressin treated wild-type and Pde1a InsA homozygous mice (upper panels); One-way ANOVA with post-hoc Tukey test was used for the statistical analysis; using a two-way ANOVA, both Pde1a genotype P

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: FIG 3 Cell-associated MUC1 levels are upregulated during IAV infection and IFN treatment. HAE were stimulated with (A) IFN-beta, (A) TNF-alpha, or (B) IFN-lambda3 or (C) infected with PR8 (5 x 10 4 PFU; approximate MOI of 1), and MUC1 expression was quantified by qPCR after 24 h of treatment. (D) HAE were stimulated as indicated or infected with PR8 as for panels A to C for 24 h, protein lysate collected, and MUC1 expression quantified by Western blotting for MUC1-CT. MUC1-CT band intensity was analyzed by densitometry relative to actin band intensity. (E) HAE were stimulated with IFN-beta, IFN-lambda3, or PR8 alone (-) or in the presence of the JAK inhibitor ruxolitinib (JAKi) or DMSO as a vehicle control (veh). Additional cultures were stimulated with Udorn or TNF-alpha alone. After 24 h, lysate was collected and analyzed by Western blotting for MUC1-CT, MX1, or actin. Results in panels A to C are from three experimental replicates utilizing three different HAE donors with a minimum of three biological replicates from each donor. The densitometry analysis (D) shows data from four experimental replicates utilizing four different HAE donors with one biological replicate from each donor. All experimental results were analyzed by the Mann-Whitney U test compared to mock conditions and are significant where indicated (*, P < 0.05; ns, not significant).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: FIG 4 Type 1 IFN and IAV broadly upregulate MUC1 expression across HAE. (A) HAE were stimulated with IFN-beta or mock stimulated (left) or infected with Udorn (5 x 10 2 PFU, approximate MOI of 0.01) (right) and fixed for immunohistochemical detection of MUC1-CT (purple), acetylated alpha-tubulin (cilium marker; green), and nuclei at the indicated time points. (B to E) HAE were infected with Udorn (5 x 10 4 PFU, approximate MOI of 1), fixed at 24 hpi, and stained en face for MUC1-CT (purple) along with (B) viral antigen (nucleoprotein, green), or (C) a ciliated cell marker (acetylated alpha-tubulin; green). The mean intensity (D) or total area staining positive for MUC1-CT (E) was quantified by FIJI on four additional cultures across two donors after infection as for panels B and C and analyzed by the Mann-Whitney U test compared to the mock condition, indicating significance (*, P < 0.05). Results in panel A are from one experimental replicate utilizing two different HAE donors (left and right) and one biological replicate from each donor. Results in panels B and C are from the same donor. Results in panels D and E are from two experimental replicates utilizing two different HAE donors with two biological replicates from each donor. Bars = 20 mum (A) and 25 mum (B and C).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: FIG 6 Establishment and characterization of immortalized HAE depleted of MUC1. Immortalized airway epithelial BCi-NS1.1 cells were transduced with CRISPR/Cas9 and sgRNA targeting (A) MUC1 exon 5 for protein depletion (MUC1 KO ) or without (control [Ctl]) the predicted targeting site. (B) Genomic DNA was extracted and used in a T7 endonuclease I cleavage assay demonstrating editing at the target site. (C) After differentiation, total HAE lysate was collected, separated by PAGE, and blotted for nontargeted tethered mucin MUC4 (extracellular domain), MUC1-ED, MUC1-CT, and actin. (D) Representative histological sections of paraffin-embedded cultures show normal ciliated epithelium. H&E counterstain. Bar = 20 mum. (E) Fluorescent microparticles were applied apically to indicated cultures to determine mucociliary transport rate. MCC between culture types was analyzed by the Mann-Whitney U test (****, P < 0.0001).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 12 MUC1 is upregulated in both tumor and immune cells in repose to AOM/DSS. MUC1 IHC staining colon section from WT mice ether treated with AOM followed by DSS as per Figure 1 , A at day 85 or untreated at day 0 (n >= 6). Statistics: box plots show median, quartiles and range. Mann-Whitney U test. * vs Day 0, ** P < .01. The data are representative of 3 independent experiments.