P stat3 try705

Cells were lysed at 4 °C in RIPA buffer supplemented with protease and phosphatase inhibitors. Equal loads of 30 μg of proteins were electrophoretically separated using SDS/polyacrylamide gels and then transferred to the PVDF membrane (Millipore, Bedford, MA, USA). After blocking with 5% nonfat milk, the membrane was made to react with specific antibodies (ALDH1A3, Abcam #ab129825, 1:1000; STAT3, Cell Signaling #4904, 1:1000; p-STAT3 (Try705), Cell Signaling #9145, 1:1000; STAT1, Santa Cruz #sc-417, 1:1000; p-STAT1 (Try701), Cell Signaling #9167, 1:1000; β-actin, Sigma-Aldrich #A2228, 1:1000) overnight at 4 °C and then incubated with horseradish peroxidase-conjugated secondary antibody for 1 h. Blots were visualized using an ECL-Plus detection kit (PerkinElmer Life Sciences, Boston, MA, USA).

Primary antibodies: SARS-CoV-2 Spike S1 Antibody (HC2001, GenScript Biotech, USA), ACE2 for Western blot (WB; sc-20998, Santa Cruz, USA), ACE2 for immunofluorescence (IF) (sc-73668, Santa Cruz, USA), STAT3 (12640, Cell Signalling, USA), p-STAT3 (Try705) (9145, Cell Signalling, USA), EGFR (sc-120, Santa Cruz, USA), p-EGFR(Tyr1068) (44788G, Thermo-Fisher Scientific, USA), p-EGFR(Tyr1086) (369700, Thermo-Fisher Scientific, USA), ERα (ab14020, Abcam, USA), β-actin (sc-8432, Santa Cruz, USA). Secondary antibodies: goat anti-human IgG Alexa Fluor 555 (A-21433, ThermoFisher, USA), goat anti mouse IgG-FITC (sc-2010, Santa Cruz, USA), goat anti-chicken IgY H&L Alexa Fluro 647 (ab150175, Abcam, USA), goat anti-mouse IgG H&L Alexa Fluro 647 (ab150115, Abcam, USA), goat anti-mouse IgG-HPR (sc-2031, Santa Cruz, USA), goat anti-rabbit IgG H&L-HRP (ab6721, Abcam, USA). Phalloidin labelled with Alexa Fluor 647 for F-actin staining (A22287, ThermoFisher, USA). Hoescht 33342 for nuclear (DNA) staining (H3570, ThermoFisher, USA).