Goat anti rabbit hrp conjugated secondary antibody

Mouse tissues were dissected and homogenized in ice-cold lysis buffer consisting of 150 mM NaCl, 50 mM Tris at pH 7.5, 1% NP-40, 0.1% SDS, 1% (vol/vol) Triton X-100, 1 mM PMSF, and 1 X protease inhibitor cocktail (Sigma-Aldrich, Saint Louis, MO). After centrifugation at 4°C, the supernatant was collected and separated by polyacrylamide gel electrophoresis (PAGE), then transferred to PVDF membrane. After blocking in PBS containing 5% BSA and 0.1% Tween-20, the membrane was incubated with rabbit anti-SNX9 polyclonal antibody (Proteintech, Cat. No. 15721-1-AP, or Sigma-Aldrich, Cat. No. HPA031410) at 4°C over night, followed by incubation with HRP-conjugated goat anti-rabbit secondary antibody (Bio-Rad, Cat. No. 170-6515) at 4°C for an hour. The signals were detected with the ECL system (Cell Signaling Technology, Danvers, MA). Then the same blot was incubated with mouse anti-GAPDH polyclonal antibody (Millipore, Cat. No. MAB374) at 4°C over night, followed by incubation with HRP-conjugated goat anti-mouse secondary antibody (Bio-Rad, Cat. No. 170-6516) at 4°C for an hour. The signals were detected with the ECL system (Cell Signaling Technology).

Protein expression was measured using western blot analysis, as described previously [41 (link)]. Briefly, proteins were separated by electrophoresis (SDS/PAGE) and transferred using a semi‐dry transfer system (Bio‐Rad, Hercules, CA, USA). This was followed by overnight incubation with listed primary antibodies at 4 °C, and subsequently with secondary antibodies for 1 h at RT. Primary antibodies against ALDH1A1 (ab52492; RRID:AB_867566), CD44 (ab51037; RRID:AB_868936), EpCAM (ab223582; RRID:AB_2762366), and PON1 (ab92466; RRID:AB_10562283) were purchased from Abcam while Vinculin (#13901; RRID:AB_2728768) and GAPDH (#5174; RRID:AB_10622025) from Cell Signaling Technology (Danvers, MA, USA). HRP‐conjugated Goat anti‐Rabbit secondary antibody from Bio‐Rad (1706515; RRID:AB_11125142) was used.

Western blot analysis was performed using cell lysates prepared in T‐PER tissue protein extraction reagent (Thermo) with protease and phosphatase inhibitor cocktail (Roch). After resolved by SDS‐PAGE (4%–20% TGX gradient gel, BioRad), proteins were transferred to PVDF membrane (Bio‐Rad), rinsed in PBST, blocked in 5% dry milk, and incubated with a rabbit monoclonal antibody against mouse β3‐Integrin (at 1:1000 dilution, #13166; Cell Signaling Technology) and a mouse monoclonal antibody for GAPDH (at 1:1000 dilution, #RDI‐TRK5G4‐6C5; Research Diagnostics Inc). HRP‐conjugated goat anti‐rabbit secondary antibody (1:3000, Bio‐Rad) and goat anti‐mouse secondary antibody (1:3000, Invitrogen) were used for detection by chemiluminescence (SuperSignal WestPico PLUS; Thermo). Signals were obtained by an LICOR‐Odyssey scanner.