Ab133411

For fluorescent immunoblotting, proteins separated by SDS-PAGE were transferred to low-fluorescence PVDF membrane (Abcam, ab133411), blotted by anti-FLAG rabbit polyclonal primary antibody (MBL, PM020, 1 : 2000) followed by goat anti-rabbit Alexa Fluor 488 conjugated secondary antibody (Thermo, A11034, 1 : 200). The membrane was scanned with a ProteinSimple FluorChem M system (excitation 475 nm/537 nm filter, 26 nm band-pass for Alexa Fluor 488 and excitation 632 nm/710 nm filter, 40 nm band-pass for Alexa Fluor 647). For chemiluminescent immunoblotting, proteins were blotted by anti-FLAG rabbit polyclonal primary antibody (1 : 2000), followed by anti-rabbit-HRP conjugated secondary antibody (1 : 2000).

pGCs were seeded in T-25 flasks at a density of 1 × 10⁵ cells/flask. pGCs were cultured in DMEM/Ham’s F-12 with 15% FBS and 1% Pen/Strep and induced in SMG for 72 h. pGC lysate was prepared with an Optiblot LDS Sample Buffer (ab119196, Abcam, Cambridge, MA, USA) and was loaded into the Precast Gel SDS-PAGE 4–12% (ab139596, Abcam, Cambridge, MA, USA). The electrophoresis was performed in an Optiblot SDS Run Buffer (ab119197, Abcam, Cambridge, MA, USA) for 2 h at 50 V. The protein was transferred to a methanol-treated PVDF membrane (ab133411, Abcam, Cambridge, MA, USA). The membrane was treated with blocking buffer (ab126587, Abcam, Cambridge, MA, USA). The membrane was incubated with primary antibodies overnight at 4 °C. Anti-Cdk4 (ab137675, Abcam, Cambridge, MA, USA), anti-Cdk6 (ab124821, Abcam, Cambridge, MA, USA), and anti-Cyclin D1 (ab40754, Abcam, Cambridge, MA, USA) were used at a 1:5000 dilution. Anti α-tubulin (ab52866, Abcam, Cambridge, MA, USA) was used at a 1:10,000 dilution. The membrane was washed three times with 1X TBST. The membrane was incubated with goat anti-rabbit IgG (HRP) (ab6721, Abcam, Cambridge, MA, USA) at room temperature for 1 h, then was washed three times with 1X TBST. The blots were visualized using a ECL Western Blotting Substrate Kit (ab65623, Abcam, Cambridge, MA, USA). Imaging was carried out with an X-ray film in a darkroom.

Kidney lysates were prepared on ice, then centrifuged for 10 min (14,000 rpm, at 4 °C). Lysate protein concentration was determined using a Protein Assay Kit I (Catalog # 5000006, Bio-Rad, Hercules, CA, USA). Then, lysate samples (50 μg protein) were separated by a 10 % Tris-Glycine gel and transferred for 2 h using a semi-dry transfer cell to a PVDF membrane (ab133411, ABCAM, Cambridge, UK). After separation, membranes were blocked with 5 % non-fat dry milk in TBST (Tris 0.01 M pH 7.4, 100 mM NaCl and 0.1 % Tween 20) for 30 min. This was followed by incubation overnight at 4 °C with one of the following primary antibodies: anti-Nrf2 (MBS714561), anti-Keap1 (MBS714561), anti–HO–1 (MBS220919) (MyBioSource, San Diego, CA, USA). Membranes were washed with TBST and incubated with HRP-conjugated secondary antibody for 1 h. Immunoreactivity was visualized using an enhanced chemiluminescence kit (GE Healthcare, Piscataway, NJ, USA). β-Actin was the reference protein using a primary antibody with catalog # ab8226 (ABCAM, Cambridge, UK).