Fig. 5

GRP78 was required for ERK phosphorylation stabilization on microtubules. A Circle diagram showing the peptides identified from immunoprecipitation and mass spectrometry (IP-MS) analysis. Proteins interacting with ERK were identified, in which microtubule-associated proteins are indicated (white circle). GRP78 was pulled down by a specific antibody or IgG as a negative control in B H460 and C A549 cells, and p-ERK, ERK, and GRP78 were evaluated by immunoblotting. Data were obtained from triplicate independent experiments. D H460 and E A549 cells were treated with or without 10 µM nocodazole (NDZ) for 1 h. Lysates were separated into soluble (S) and pellet (P) fractions using a microtubule sedimentation protocol and analyzed for GRP78 and α-tubulin by immunoblotting. The ratio of the pellet to the total fraction was calculated. Data are presented as the mean ± SEM. *p < 0.05 vs. control cells (n = 3). F H460 and A549 cells were transfected with various concentrations of GRP78 siRNA (siGRP78) or control siRNA (siCtrl). The expression levels of GRP78, p-ERK, and ERK were analyzed by immunoblotting. The intensity was normalized to that of GAPDH. Data are presented as the mean ± SEM. *p < 0.05 vs. siCtrl cells (n = 3). (G) GRP78 knockdown (siGRP78) and control (siCtrl) H460 and A549 cells were lysed, separated into soluble (S) and pellet (P) fractions using a microtubule sedimentation protocol, and analyzed for p-ERK and α-tubulin by immunoblotting. The ratio of the pellet to the total fraction was calculated. Data are presented as the mean ± SEM. *p < 0.05 vs. siCtrl cells (n = 3)