Anti stc1

Equal amounts of protein were separated by 10% SDS–PAGE and then transferred to polyvinylidene fluoride membranes. After incubation with the primary antibodies and secondary antibody, the blots were analyzed using enhanced chemiluminescence detection system. The primary antibodies used were anti-STC1 (1:1,000, Santa Cruz, Shanghai, China), anti-VEGFA (1:1,000; Abcam, Shanghai, China), anti-VEGFR2, anti-AKT, anti-phosphorylated (p)-AKT, anti-mTOR, and anti-p-mTOR (1:1,000; Cell Signaling Technology, Shanghai, China).

Expression and subcellular localization of AP1 and of phosphorylated JNK (p-JNK) were tested by indirect immunofluorescence staining and confocal microscopy. Expression of STC1 was assessed by immunohistochemistry. Staining was done on FFPE tissue sections or cultured cells grown in slide dishes and fixed with cold methanol. The antibodies used were anti-phospho-JNK (Cell signaling), anti-AP1 (Sigma), and anti-STC1 (Santa Cruz). Primary antibodies were diluted according to manufacturers’ recommendations and incubated at 4°C overnight. Secondary antibodies labeled with Alexa Fluor 555 and Alexa Fluor 488 (Invitrogen) or HRP (Dako), were incubated for one hour at room temperature. For FFPE tissue sections the nonspecific background was blocked using blocking solution (Dako) and the staining was assessed qualitatively and scored as positive or negative. Positive score is given only when more than 10% of tumor cells show non-ambiguous staining. Staining of immune cells or any infiltrating cells other than the tumor follicular cells with the anti-p-JNK or anti-STC1 antibodies was not considered as positivity.