Anti gpnmb

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.