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

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

Journal: Cell reports

Article Title: Proteogenomic characterization of primary colorectal cancer and metastatic progression identifies proteome-based subtypes and signatures

doi: 10.1016/j.celrep.2024.113810

Figure Lengend Snippet: (A) A heatmap with immune signature and clinical attributes. The immune score significantly correlates with IHC immune infiltrate scores and immune-related pathways, except for CD4 and FOXP3 IHC scores. * denotes statistical significance. (B) A GSE plot with antigen processing and presenting pathways from KEGG and REACTOME terms. Both antigen processing pathways are strongly downregulated in the immune-cold group. (C) A correlation plot (Spearman correlation coefficient) of IHC scores that involve the antigen processing machinery. Protein expression of STAT is derived from mass spectrometry data. The IFNγ response score is derived from ssGSEA analysis with HALLMARK_INTERFERON_GAMMA_RESPONSE. *p < 0.05, **p < 0.01, ***p < 0.001. Clear correlations of CD4-MHC class II and CD8-MHC class II are shown with statistical significance. In addition, the IFNγ pathway and STAT1 positively correlate with their direct targets (IRF1 and CIITA) and with downstream targets (MHC class I and II). (D) A correlation plot (Spearman correlation coefficient) with IRF1 IHC score and antigen processing machinery protein expression. *p < 0.05, **p < 0.01. ***p < 0.001. IRF1 expression positively correlates with immunoproteasome subunits but negatively correlates with catalytic conventional proteasome subunits. (E) Boxplots of IHC analysis results for HLA-ABC, HLA-DP/DQ/DR, CD3, CD4, CD8, IFR1, and CIITA. 22 P1, 42 P2, 51 P3, 16 M1, 53 M2, and 30 M3 samples were assessed. M1 shows the lowest expression of HLA-ABC (MHC class I) among all proteome subtypes. Although the infiltrating lymphocyte profile (CD3, CD4, and CD8) shows no significant differences, M1 has the lowest CD3+ lymphocyte count and lower median counts of CD8+ lymphocytes than other subtypes. This is concordant with the lowest expression of HLA-ABC (MHC class I) in the M1 subtype. In addition, IRF1, a key transcription initiator of MHC class I, shows the lowest expression in M1. (F) Kaplan-Meier survival curve analysis stratified by CD4 expression level of pCRC. (G) Kaplan-Meier survival curve analysis stratified by CD8 expression level of pCRC. (H) Kaplan-Meier survival curve analysis stratified by CD4 expression level of mCRC. (I) Kaplan-Meier survival curve analysis stratified by CD8 expression level of mCRC.

Article Snippet: Rabbit monoclonal anti-HLA class I ABC , Abcam , Cat. # ab225636; RRID: N/A.

Techniques: Expressing, Derivative Assay, Mass Spectrometry