Effect of Clast1/LR8 on the Ras signaling pathway. (a) RT‐PCR analysis of Clast1/LR8 expression in NIH3T3/vector cells, H‐RasV12 NIH3T3 cells, and H‐RasV12 NIH3T3 cells transfected with a Clast1/LR8 expression vector. (b) Western blot analysis for Clast1/LR8 (X‐press) in H‐RasV12 NIH3T3 cells transfected with a Clast1/LR8 expression vector or empty vector. (c) The indicated cells were plated in DMEM supplemented with 5% FBS, washed twice in PBS 48 h later, then incubated in serum‐free medium for 24 h. Cells were then stimulated with or without 20% FBS for 15 and 30 min. Phosphorylated ERK1/2 (pERK1/2) and Akt (pAkt) levels, as well as total ERK1/2 and Akt protein levels, were analyzed by Western blotting.