Anti hsf1

Proteins were transferred to PVDF membrane, blocked in blocking buffer (5% w/v skim milk powder in PBS-T) for 1 h at room temperature and then incubated with primary antibody in blocking buffer. The following antibodies were incubated overnight at 4 °C: anti-HSPA1A (Origene, cat #TA500772, 1:10,000), anti-HSF1 (Abcam, cat #ab52757, 1:40,000), anti-BAG1 (Abcam, cat #ab32109, 1:750), and anti-myc (Thermofisher, cat #13–2500, 1:1,000). The blots were washed in PBS-T and then incubated with either anti-rabbit secondary antibody (Invitrogen, cat #65-6120, 1:20,000) or anti-mouse secondary antibody (Invitrogen, cat #31430, 1:20,000) in PBS-T for 1 h at room temperature. Proteins were detected by an enhanced chemiluminescence kit (Clarity, BioRad). The uncropped blots are shown in Supplementary Fig. 7.

Protein was extracted from AGS cells after indicated treatments. After calculating protein concentration, equal amounts of the SDS-PAGE-separated lysates were transferred onto polyvinylidene difluoride (PVDF) membranes, which were subsequently blocked at room temperature with 1 × TBS containing 0.1% Tween 20 and 5% skim milk. The membranes were incubated at 4 °C overnight with the following primary antibodies: anti-caspase-3, anti-caspase-8, anti-caspase-9, anti-survivin, anti-HSP27, anti-HSP70, anti-HSP90, anti-p-ERK (Thr202/Tyr204), anti-ERK, anti-p-p38 (Thr180/Tyr182), anti-p38, anti-p-JNK (Thr183/Tyr185), anti-JNK (Cell Signaling Technology, Danvers, MA, USA), anti-β-actin, anti- Bcl-xL, anti-Bcl-2, anti-cyclin B1, anti-cyclin D1, anti-MMP9, anti-MMP2, anti-VEGF (Santa Cruz Biotechnology, Inc., Dallas, TEX, USA), anti-HSF1, anti-pHSF1 (Abcam, Inc., Waltham, MA, USA), and anti-cleaved caspase (Genetex, Irvine, CA, USA). The membranes were washed three times before exposure to diluted anti-rabbit or anti-mouse IgG secondary antibodies (Santa Cruz Biotechnology, Inc.) for an hour at room temperature. The blots were washed thrice with 1 × TBS-T buffer for 10 min between each stage. The membranes were identified using enhanced chemiluminescence (Millipore, Billerica, MA, USA).