ERK-specific gene silencing in NIH 3T3 cells differentially affects MEK-ERK interactions. (a) ERK1- and ERK2-specific NIH 3T3 clones with stable shRNA expression only (left) or also co-transfected with H-RasQ61L (right) were isolated and checked for ERK expression levels by western blot analysis, as in Figure 1. Two clones (I and II) for each transfection are shown. (b) Lysates from wild-type NIH 3T3 control, ERK1 KD and ERK2 KD clones growing in 10% serum were incubated with anti-MEK-1/2 polyclonal antibody. Immune complexes (IP) were resolved in SDS-PAGE and western blotted (WB) with polyclonal anti-ERK1 (sc-94, top) and anti-ERK2 (sc-153, bottom) antibodies. (c) Bands from (b) were quantified and the relative fold increase in ERK1 and ERK2 levels in the knockdown samples over the corresponding wild-type controls were calculated (only samples probed with anti-ERK antibody sc-94 are indicated). Data are representative of three independent experiments, expressed as mean ± SEM.