Anti hsf1

Total proteins of the kidney cortex were extracted RIPA lysis buffer (Beyotime, China), and the concentration was measured by the BCA method. The samples were transferred to PVDF membrane, blocked with 5% BSA at room temperature for 1 hour, and the PVDF membrane was incubated with the corresponding antibodies including anti-HSF1 (1 : 500, Santa cruz (cat# sc-8402), USA)/RELA (1 : 500, Santa cruz (cat# sc-17757), USA) at 4°C overnight. Furthermore, the membranes were incubated with the corresponding HRP-conjugated secondary antibody after rinsing and exposed with ECL Chemiluminescence Kit (Thermo, Waltham, MA, USA). Image J software was conducted to analyze the gray value, and the protein levels were corrected for the housekeeping gene GAPDH.

For western blot analysis, solei were homogenized in reducing sample buffer (80 mM Tris-HCl, pH6.8, 2% sodium dodecyl sulfate, 10% glycerol and 0.1 M dithiothreitol) supplemented with Halt protease and phosphatase inhibitor cocktail (ThermoFisher Scientific). The following antibodies were used for western blot analysis: anti-HSF-1 (Santa Cruz Biotechnology), anti-hsp70 (StressMarq), anti-hsp90 (BD Transduction Laboratories), anti-P-CaMKII (ThermoFisher Scientific), anti-CaMKIIβ (Invitrogen). The quantitative analysis was performed using ImageJ software.

Nuclear or cytoplasmic extracts were prepared from AGS cells after galectin-3 siRNA and HSF-1 siRNA treatments. These experiments have been described in a previous study [46 (link)]. Western blot analysis was carried out using the methodology from a previous study [47 ]. Briefly, cells were lysed in RIPA buffer (Biosesang, Korea) containing a protease inhibitor cocktail (Sigma, USA). The primary antibodies were: anti-β-actin, anti-galectin-3, anti-neogenin-1, anti-HSF-1, and anti-HSP70 from Santa Cruz, and anti-ROCK-1 from Cell Signaling, also, anti-pROCK-1(T455/S456) antibody from Bioss. Proteins of interest were detected using ECL solutions (Amersham Life Science) with an LAS-3000 (Fujifilm) detector according to the manufacturer's directions.

Immunoblot analysis for U2OS cells was performed as described (61 (link)) using the following antibodies: anti-BMAL1 (14020, Cell Signaling, Danvers, MA, USA), anti-CLOCK (sc-6927, Santa Cruz Biotechnology, Dallas, TX, USA), anti-PER2 (20359-1-AP, Proteintech, Chicago, IL, USA), CRY1 (A302-614A, Bethyl Laboratories, Montgomery, TX, USA), anti-CRY2 (13997-1-AP, Proteintech), anti–REV-ERBα (13418, Cell Signaling), anti–REV-ERBβ (GTX115322, GeneTex, Irvine, CA, USA), anti-CCNB1 (#4138, Cell Signaling), anti-CCND1 (ab134175, Abcam, Cambridge, MA, USA), anti-CDK4 (12790, Cell Signaling), anti-AKT (4685S, Cell Signaling), anti-phospho AKT Ser⁴⁷³ (pAKT) (4060S, Cell Signaling), anti-p44/42 mitogen-activated protein kinase (MAPK) (Erk1/2) (4695S, Cell Signaling), anti–phospho-p44/42 MAPK (Erk1/2)-Thr²⁰²/Tyr²⁰⁴ (pERK) (4370S, Cell Signaling), anti–caspase-3 (9662S, Cell Signaling), anti-HSF1 (sc-17756, Santa Cruz Biotechnology), anti-HSP70 (4872S, Cell Signaling), anti-HSP90AA1 (8165S, Cell Signaling), anti-HSP90AB (7411S, Cell Signaling), anti-HSP90B1 (Grp94) (2104S, Cell Signaling), and anti-TRAP1 antibody (GTX102017, GeneTex). Anti-GAPDH (glyceraldehyde-3-phosphate dehydrogenase) (sc25778, Santa Cruz Biotechnology), anti-tubulin (ab18251, Abcam), and anti–β-actin (4967S, Cell Signaling) were used as loading control antibodies.

The modulatory effects of compounds 1 and 2 on HSF1 and the HSPs’ expression were evaluated using a previously established protocol [21 (link)]. Proteins in lysates were separated by SDS-PAGE, electrotransferred to nitrocellulose membranes (GE Healthcare, Amershan, UK), subsequently blotted with specific antibodies, and visualized using an ECL detection system (Thermo Scientific, Waltham, MA, USA). Anti-HSF1, anti-Hsp27, anti-Hsp70, and β-actin antibodies were purchased from Santa Cruz Biotechnology (California, CA, USA). All of the results represent the mean ± SD of three independent experiments performed in triplicate 24 h after treatment. Furthermore, p-values < 0.05 were considered statistically significant, comparing the quantitative values of HSP70, HSP27, or HSF1 expression levels between treated and untreated control cells.