resource source identifier antibodies hla class i abc rabbit polyclonal antibody proteintech  (Proteintech)

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: The aryl hydrocarbon receptor (AHR) drives human leukocyte antigen (HLA)-II expression in human melanoma

doi: 10.1186/s13046-026-03673-y

Figure Lengend Snippet: AHR and ARNT regulate basal HLA-II expression on cancer cells. A Western blot analysis confirming AHR or ARNT overexpression (OE) in A375, WM115, and SKMEL2 melanoma cell lines. Protein levels of AHR, ARNT, and HLA-DRA were assessed using anti-AHR (clone D5S6H), anti-ARNT (clone D28F3), and polyclonal anti–HLA-DRA antibodies, respectively, with β-tubulin detected using an anti–β-tubulin antibody (clone C66) as a loading control. B- C Flow cytometry analysis of surface pan–HLA-II expression in A375, WM115, and SKMEL2 cells overexpressing AHR or ARNT. Representative histograms from three independent experiments show pan–HLA-II expression, with dashed lines indicating the median of the empty vector control group ( B ). The gMFI was quantified across three independent experiments, normalized to the empty vector control group, and presented as fold change ( C ). D Western blot analysis of AHR, ARNT, and HLA-DRA protein levels in AHR or ARNT reconstituted A375 cells generated by reintroducing AHR or ARNT into respective KO cells. Protein levels of AHR, ARNT, and HLA-DRA were assessed using anti-AHR (clone D5S6H), anti-ARNT (clone D28F3), and polyclonal anti–HLA-DRA antibodies, respectively, with β-tubulin detected using an anti–β-tubulin antibody (clone C66) as a loading control. E - F Flow cytometry analysis of surface pan–HLA-II expression in AHR- or ARNT-reconstituted A375 cells. Representative histograms from three independent experiments show pan–HLA-II expression ( E ). The gMFI was quantified across three independent experiments, normalized to the NTC group, and presented as fold change ( F ). Surface pan–HLA-II expression was detected using APC anti-human HLA-DR, DP, DQ Antibody (clone Tü39) ( B - C and E - F ). Data are represented as mean ± SD ( C and F ). Statistical analysis by one-way ANOVA ( C ) and unpaired Student’s t-test ( F ); * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. vec, empty vector control

Article Snippet: The following primary antibodies were used: AHR Rabbit mAb (clone D5S6H, CST #83200, RRID: AB_2800011), ARNT Rabbit mAb (clone D28F3, CST #5537, RRID: AB_10694232), HLA-DRA polyclonal Rabbit Ab (Boster #A01195), β-Tubulin Mouse mAb (clone C66, Abmart # M20005 , RRID: AB_2920648), and β-Actin Rabbit mAb (clone 13E5, CST #4970, RRID: AB_2223172).

Techniques: Expressing, Western Blot, Over Expression, Control, Flow Cytometry, Plasmid Preparation, Generated

Journal: Cell Reports Medicine

Article Title: Cathepsin-D-mediated MHC class I degradation contributes to immune evasion in colorectal cancer

doi: 10.1016/j.xcrm.2025.102534

Figure Lengend Snippet: CTSD impairs antigen presentation and CD8 T cell activation (A) The expression of MHC class I on the surface of MC38 cells was analyzed by flow cytometry. (B) DLD1 and SW480 stable cells were subjected to western blotting for indicated protein. (C) Class I HLA expression levels were assessed by flow cytometry in human CRC cells following 24-h stimulation with IFN-γ. (D) A schematic representation of the in vitro coculture assay involving tumor and immune cells. (E) In vitro cytotoxicity assays for activated OT-1 CD8 + T cells against CTSD knockdown or control MC38-OVA cells. (F and G) Post-coculture of tumor cells with OT-1 splenocytes, the percentages of GZMB + and IFN-γ + cells within the CD8 + T cell population were determined by flow cytometry. Three independent repeated experiments. (H) OT-1 cells were co-cultured with MC38-OVA cells in the presence of H-2K b -SIINFEKL or IgG antibodies for 48 h, and OT-1 cell proliferation was measured by CFSE dilution. (I and J) Tumor volumes and Kaplan-Meier survival curves were analyzed in subcutaneous tumor-bearing mice inoculated with NC, CTSD knockdown, H2K1 knockout, and CTSD knockdown plus H2K1 knockout CT26 cells ( n = 6). (K) Western blotting analyses of HLA-ABC and CTSD expression in CRC cell lines. Right, quantitative estimates of HLA-ABC and CTSD levels based on western blotting analyses. (L) Immunohistochemical staining of CTSD and HLA-ABC in CRC tumors. Pearson’s correlation test was used to analyze correlations between the H-scores of CTSD and HLA-ABC in CRC tumors. (M) Kaplan-Meier survival curves for overall survival based on the expression status of CTSD hi /HLA-ABC lo and CTSD lo /HLA-ABC hi . (N) Negative correlation of CTSD mRNA levels with that of CD8A in ESCA, LIHC, and OV. Data are means ± SEM. p values were determined by Student’s t test (A, C, F, G, and H), two-way ANOVA (E and I), log rank test (J and M), and Pearson’s correlation test (K, L, and N). n.s represents no significance.

Article Snippet: Finally, the staining was visualized using 3,3′-Diaminobenzidine (DAB) for 30 s. CTSD rabbit antibody (ab75852, Abcam; 1:200 dilution), HLA-ABC rabbit antibody (15240-1-AP, ProteinTech; 1:1,000 dilution) were used for immunostaining.

Techniques: Immunopeptidomics, Activation Assay, Expressing, Flow Cytometry, Western Blot, In Vitro, Co-culture Assay, Knockdown, Control, Cell Culture, Knock-Out, Immunohistochemical staining, Staining

Journal: Cell Reports Medicine

Article Title: Cathepsin-D-mediated MHC class I degradation contributes to immune evasion in colorectal cancer

doi: 10.1016/j.xcrm.2025.102534

Figure Lengend Snippet: CTSD promotes lysosome-mediated degradation of MHC class I in tumor cells (A and B) Western blotting analyses of HLA-ABC expression in CTSD-knockout SW480 cells treated with cycloheximide (CHX) (A) or bafilomycin A1 (Baf A1) (B). Right, quantitative estimates of HLA-ABC levels based on western blotting analyses. (C and D) Domain structure of mouse CTSD (C). SP, signal peptide. Immunoprecipitation/western blotting analyses of the interaction between full-length H2-K1-HA and full-length or partially deleted mouse CTSD-Flag using anti-flag antibody or anti-HA antibody (D). (E and F) Domain structure of H2K1 (E). SP, signal peptide. Immunoprecipitation/western blotting analyses of the interaction between full-length mouse CTSD-Flag and full-length or partially deleted H2-K1-HA using anti-flag antibody or anti-HA antibody (F). (G and H) BLAB/c mice were injected with CTSD knockdown CT26 cells, with re-expressed CTSD-WT or CTSD-D1. Tumor volume (G), representative tumor images, and tumor weight (H) were shown ( n = 6). (I) Knockout CTSD promotes HLA-ABC recycle to membrane. Representative fluorescence confocal images of endogenous HLA-ABC distribution in CTSD-knockout HCT116 cells. Scale bars, 10 μm. WGA, wheat germ agglutinin (a marker of plasma membranes). (J and K) Left, western blotting analyses of HLA-ABC in the plasma membrane (J) and lysosome (K) fractions of CTSD knockout cells. Right, quantitative estimates of MHC class I (HLA-ABC) expression levels, based on data from the left panels. Three independent repeated experiments. Data are means ± SEM. p values were determined by two-way ANOVA (G) and one-way ANOVA (H). n.s represents no significance.

Article Snippet: Finally, the staining was visualized using 3,3′-Diaminobenzidine (DAB) for 30 s. CTSD rabbit antibody (ab75852, Abcam; 1:200 dilution), HLA-ABC rabbit antibody (15240-1-AP, ProteinTech; 1:1,000 dilution) were used for immunostaining.

Techniques: Western Blot, Expressing, Knock-Out, Immunoprecipitation, Injection, Knockdown, Membrane, Fluorescence, Marker, Clinical Proteomics

Journal: Cell Reports Medicine

Article Title: Cathepsin-D-mediated MHC class I degradation contributes to immune evasion in colorectal cancer

doi: 10.1016/j.xcrm.2025.102534

Figure Lengend Snippet: Inhibition of CTSD with pepstatin A impedes immune evasion and increases the sensitivity of anti-PD-1 antibody (A) Western blotting analyses of HLA-ABC expression in DLD1 and SW480 cells treated with pepstatin A (Pep A). (B) Flow cytometry analysis was used to detect the expression of MHC class I on the surface of MC38 cells treated with Pep A. (C) Representative fluorescence confocal images of endogenous HLA-ABC distribution in HCT116 cells treatment with Pep A. Scale bars, 10 μm. (D and E) Left, western blotting analyses of HLA-ABC in the plasma membrane (D) and lysosome (E) fractions of HCT116 cells treatment with Pep A. Right, quantitative estimates of MHC class I (HLA-ABC) expression levels, based on data from the left panels. Three independent repeated experiments. (F) A schematic representation of AOM/DSS-induced spontaneous colon cancer mouse models in BALB/c mice. (G) Representative gross images of the colons at day 100 for the indicated groups of mice. (H) Tumor number in the indicated groups of mice from (G) ( n = 8). (I) Representative images of bioluminescent imaging of indicated groups in CT26 cecal subserosal model on days 5 and 25. (J) Relative fluorescence intensity fold change by bioluminescent imaging in the indicated groups of mice from I ( n = 6). (K and L) BALB/c mice bearing subcutaneous CT26 tumor cells received PBS, Pep A, anti-PD-1 antibody, or Pep A plus anti-PD-1 antibody therapy. Tumor volumes (K) and Kaplan-Meier survival curves (L) on the indicated days after inoculation with tumor cells ( n = 6). (M) Immunofluorescent staining of CD8 in CT26 tumors from (K) and quantification of CD8 signals ( n = 6). Scale bar, 50 μm. (N) Primary tumors from mice were harvested for flow cytometry to determine the percentages of GZMB + and IFN-γ + cells among CD8 + T cells ( n = 6). Data are means ± SEM. p values were determined by Student’s t test (B, D, and E), one-way ANOVA (H, J, M, and N), two-way ANOVA for tumor volume (K), and log rank test (L).

Article Snippet: Finally, the staining was visualized using 3,3′-Diaminobenzidine (DAB) for 30 s. CTSD rabbit antibody (ab75852, Abcam; 1:200 dilution), HLA-ABC rabbit antibody (15240-1-AP, ProteinTech; 1:1,000 dilution) were used for immunostaining.

Techniques: Inhibition, Western Blot, Expressing, Flow Cytometry, Fluorescence, Clinical Proteomics, Membrane, Imaging, Staining