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. 2016 Jul 27;64(11):1938–1961. doi: 10.1002/glia.23034

Figure 4.

Figure 4

Recognition by 9F5 of GPNMB protein in rat GpnmbcDNA‐transfected COS‐7 cells. A–C: COS‐7 cells were transfected with pcDNA3.1‐Gpnmb or a pcDNA3.1 empty vector for 72 hr. A: Cells were double‐stained with 9F5 and a commercially available goat anti‐mouse GPNMB polyclonal antibody. d, h: Phase‐contrast images. B: Cell lysates were from COS‐7 cells transfected with pcDNA3.1‐Gpnmb (lane 1) or pcDNA3.1 empty vector (lane 2) or were from type 1 MG (lane 3). Rat GPNMB proteins were cross‐reacted with a goat anti‐mouse GPNMB antibody. Arrows indicate specific staining. C: Lysates of cells transfected with pcDNA3.1‐Gpnmb or pcDNA3.1 empty vector were immunoprecipitated by using 9F5 (under DTT (−) condition) and then immunoblotted (IB) with the anti‐GPNMB antibody (under DTT (+) condition). Lysates from rat type 1 MG was used as a positive control (P.C.) for immunoblotting with anti‐GPNMB antibody. Lower panel shows the IgG (9F5 or control IgG) that was eluted from Protein G column by SDS‐PAGE sampling buffer without DTT. D: WB analysis of rat type 1 MG reactions with 9F5 and anti‐GPNMB antibody. E: Lysates of rat type 1 MG were immunoprecipitated via anti‐GPNMB antibody and then immunoblotted by using 9F5. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]