Immobilon western chemilum hrp substrate

For dot blot analysis, TRIzol reagent was used to isolate and purify total RNA. The total RNA was denatured under UV light and cross-linked with the optimized Amersham Hybond-N⁺ membrane. After cross-linking, the membrane was blocked with 5% skim milk, and incubated with anti-m⁶A antibody at 4 °C overnight. Immobilon Western Chemilum HRP substrate (Merck Millipore) was used for visualization. For ELISA, the EpiQuik m6A RNA Methylation Quantitative ELISA Kit (P-9005-96, Epigentek) was used according to the manufacturer’s instructions.

The PC cells were lysed with radioimmunoprecipitation assay lysis buffer and protein was extracted as described previously^{15 (link)}; the protein concentrations were quantified using a DC protein assay kit (Bio-Rad, USA). The proteins were subjected to 8–12% SDS-polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane (Bio-Rad, USA)^{17 (link)}. The membranes were then blocked with 5% non-fat milk in Tris-buffered saline containing 0.1% Tween (TBST) for 2 h at room temperature and incubated with primary antibodies overnight at 4 °C. After washing with TBST (15 min) three times, the membranes were incubated with horseradish peroxidase (HRP)-conjugated anti-mouse or anti-rabbit IgG secondary antibodies for 2 h at room temperature. The membranes were washed again and the protein expression levels were visualized with the Immobilon Western Chemilum HRP substrate (Merck Millipore, Darmstadt, Germany) using an enhanced chemiluminescence detection system. The primary antibodies against CXCL11 and vimentin were obtained from Abcam (Cambridge, MA, USA); the primary antibodies against E-cadherin and N-cadherin were obtained from Proteintech (Han Wu, China). Each blotting was repeated independently three times.

Cells were harvested using a cell scraper and suspended in RIPA buffer (150 mM NaCl; 5 mM EDTA, 50 mM Tris, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS) supplemented with protease inhibitor (Roche) and phosphatase inhibitor (Roche). Endogenous proteins from whole cell extracts were isolated by sonication followed by centrifugation. Proteins were quantified using BCA protein assay (ThermoFisher Scientific) and equal amounts of proteins were loaded onto 4–12% SDS–polyacrylamide gel electrophoresis (SDS–PAGE) gels and electrophoretically transferred to a PDVF membrane using Novex iBlot transfer stack (ThermoFisher Scientific) on a iBlot gel transfer device (ThermoFisher Scientific). The membrane containing the transferred protein was blocked with 5% BSA at room temperature for 1 h. Target proteins were detected by incubating the membrane at 4 °C overnight with primary anti-Cdt1 antibody (#8064 Cell Signaling Technology) (1:500) or anti-Cdc6 antibody (#3387 cell signaling technology) (1:500) and primary anti-β-actin antibody (Santa Cruz) (1:5000). Blot was further developed using horseradish peroxidase (HRP)-conjugated secondary antibodies (1:5000) (Santa Cruz) and detected using Immobilon Western Chemilum HRP substrate (Merck). Blots were visualized on a ChemiDoc Touch Imaging System (BioRad).

Cells were washed with PBS and lysed with M-PER™ Mammalian Protein Extraction Reagent (Thermofisher Scientific, Rockford, IL, USA) supplemented with 1× protease inhibitor cocktail (Merck Millipore, Cat No. 539131, San Diego, CA, USA), while retina-tissue samples were homogenized and lysed with a RIPA lysis buffer (Beyotime, Shanghai, China) with protease inhibitor cocktail, followed by centrifugation and supernatant collection. A BCA protein assay kit (Thermofisher Scientific, Rockford, IL, USA) was used to detect the concentration of total proteins. About 10 μg of total proteins was loaded and separated by 10% SDS-PAGE gels, followed by western blotting using antibodies against Nrf2 (Abcam, ab137550, Cambridge, MA, USA), HO-1 (Abcam, ab13248, Cambridge, MA, USA), Keap-1 (Cell Signaling, 8047s, Danvers, MA, USA), and α-Tubulin (Cell Signaling, 2125s, Danvers, MA, USA). After the respective incubation of the secondary antibodies, blottings were exposed with immobilon western chemilum HRP substrate (Merck Millipore, San Diego, CA, USA) and captured using a Bio-Rad ChemiDoc XRS+ imaging system (Bio-Rad Laboratories, Hercules, CA, USA).

After SDS-PAGE, proteins were electroblotted onto a Hybond-ECL membrane (GE Healthcare, Buckinghamshire, UK), which was blocked in Tris-buffered saline containing 0.1% Tween-20 (TBS-T) and 5% skimmed milk. Proteins on the membrane were probed with antibody in blocking buffer and then the membrane was washed with TBS-T. Rabbit polyclonal anti-AcK antibody (ImmuneChem, Burnaby, BC, Canada) and Anti-His-tag mAb-HRP-DirecT (Medical & Biological Laboratories, Aichi, Japan) were used at a 1/3000 and 1/10,000 dilution respectively as primary antibodies. Anti-Rabbit IgG HRP conjugate (Promega, Madison, WI, USA) was used as the secondary antibody. Proteins on the membrane were probed with the primary antibody in blocking buffer and then washed with TBS-T. Proteins were detected by Immobilon Western Chemilum HRP substrate (Merck Millipore) and visualized on a LAS 4000 (GE Healthcare).

Cells were washed twice with cold PBS and collected by 1000 g centrifugation at 4°C for 5 min and then lysed using M-PER™ Mammalian Protein Extraction Reagent (Thermo Fisher Scientific, 78501) supplemented with protease inhibitor cocktail (Targetmol, C0001) and phosphatase inhibitor cocktail (Targetmol, C0004). After centrifugation at 12,000 g at 4°C for 10 min, the supernatant was collected. Protein concentrations were determined using a BCA assay kit (Thermo Fisher Scientific, 23225). Then, samples were separated with 10% SDS-PAGE gels and transferred to the PVDF membrane (Roche, 3010040001) followed by blocking with 5% skim milk (Wako, 190-12865). For immunoblotting, the following antibodies were adopted: NR4A3 (Santa Cruz, sc-393902), SMARCB1 (Abcam, ab222519), actin (Arigo, arg62346), GAPDH (Arigo, arg10112), NRF2 (Abcam, ab137550), goat anti-rabbit IgG antibody (HRP) (Arigo, arg65351), and goat anti-mouse IgG antibody (HRP) (Arigo, arg65350). Results were visualized on a ChemiDoc XRS+ system (Bio-Rad Laboratories, USA) with Immobilon Western Chemilum HRP Substrate (Merck Millipore, WBKLS0500).