Anti hmb 45

The following murine mAbs were used for IHC: W6/32 to detect HLA class I antigens (Dianova); bbm.1 to stain for β2m (kindly provided by G. Moldenhauer, German Cancer Research Center, Heidelberg, Germany); anti–HMB-45 (Dako) to detect melanoma cells; anti-CD3 (BD Pharmingen) to stain for T cells.
For flow cytometry, the mouse mAbs were as follows: anti–HLA-ABC-APC (eBiosciences), anti–CD54-PE and anti–HLA-DR-PECy7 (Beckmann Coulter), anti–PD-L1-PE and anti–PD-L2 (Biolegend), anti–B7-H3 (R&D Systems), anti–B7-H4 (eBiosciences); L243 was used for detection of HLA-DR molecules (17 (link)) and HC10 for labelling of β₂m-free HLA heavy chains (18 (link), 19 (link)).
The following antibodies were used for Western blot analysis: mouse anti–Melan-A/MART-1 (Zytomed), mouse anti-Tyrosinase and anti-MITF (Santa Cruz Biotechnology), rabbit anti-DCT/TRP2 (kindly provided by V. Hearing, National Cancer Institute, NIH, Bethesda); mouse anti-STAT1, rabbit anti-phospho(p)STAT1, rabbit anti-JAK1, rabbit anti-GAPDH (Cell Signaling Technology); rabbit anti-IRF1 and mouse anti-β2m (Santa Cruz Biotechnology); rabbit anti-β2m (Sigma); mouse anti-TAP1 (NOB-1) and mouse anti-tapasin (TO-3; ref. 20 (link)); mouse mAb HC10 (18 (link), 19 (link)).

Uteri and ELT3 cells were homogenized in RIPA (Santa Cruz, CA, USA). Western blots used 1:1000 rabbit polyclonal anti-phospho-S6 (Ser 235/236), anti-S6, anti-phospho-4EBP1 (Thr37/46), anti-4EBP1, anti-phospho-p44/42-ERK1/2 (T202/Y204), p44/42-ERK1/2, and 1:5000 anti-glyceraldehyde-3-phosphate-dehydrogenase (GAPDH; Cell Signaling) antibodies. IHC was performed, as described in Prizant et al. (2013) (link). Sections were incubated with 1:1000 rabbit anti-SMA (Epitomics, Burlingame, CA, USA), 1:400 anti-phospho-S6 (Ser 235/236), 1:100 anti-MMP-9, 1:300 anti-MITF, 1:500 anti-GPNMB (Sigma-Aldrich, for mouse), 1:100 anti-GPNMB (R&D Systems, for human) (Hoashi et al. 2010 (link), Rose et al. 2010b), or 1:50 anti-HMB-45 (Dako) primary antibody. For immunofluorescence, sections were incubated with 1:200 fluorescein-conjugated anti-rabbit antibody (Vector Laboratories, Burlingame, CA, USA) for 1 h at room temperature. Nuclei were labeled using 1 mg/mL Hoechst-33258 (Invitrogen). For immunofluorescence, cells were grown in poly-d-lysine pre-coated glass bottom dishes (MatTech Corporation, Ashland, MA, USA), fixed in 4% paraformaldehyde, and stained with 1:250 anti-MITF or 1:300 anti-GPNMB (Sigma-Aldrich) primary antibody.