Total protein extraction buffer

Total protein from the cells was extracted using a total protein extraction buffer (Beyotime, China). 10% sodium dodecyl sulfate polyacrylamide gel was used to isolate the proteins. After being transferred to nitrocellulose membrane, the band was blocked with 5% nonfat milk. The blots were incubated with primary antibodies and second antibodies, respectively. The antibodies and reagents were used as follows: HMGB2 (Abcam, Ab124670); JNK1/2 (CST, #9252); P-JNK1/2 (CST, #4668); Cleaved caspase3 (CST, #9661); Cleaved PARP (CST, #9545); NF-κB P65 (Abcam, Ab16502); GAPDH (CST, #5174); H3 (CST, #4499); HRP-labeled Goat Anti-Rabbit IgG (Beyotime, A0208). The results were used to visualize proteins by the enhanced chemiluminescence reagents (Tanon, China).

Total cellular protein and nuclear-cytosol protein were extracted using a total protein extraction buffer (Beyotime, China). Cell lysates were separated by SDS-PAGE followed by blocking in 1% BSA (Bovine Serum Albumin), then incubated with primary antibodies and species-specific secondary anti-bodies. Bound secondary antibodies were detected with the Odyssey imaging system (LI-COR Biosciences, Lincoln, NE). Primary antibodies used for PUF60, β-actin, PABPC1 detection were shown in Supporting Table 2.

Total protein of tissue were extracted using a total protein extraction buffer (Beyotime, China). We performed western blotting according to the traditional western blot assay method. β-ACTIN was utilized as an internal control, Antibodies (1:1000) against VDR was purchased from Servicebio (Wuhan, China).

Cell lysate was prepared using a total protein extraction buffer (Beyotime, China) and protein concentration was measured using a BCA Protein Assay Kit (Thermo Fisher Scientific, MA) as previously described.15 Equal amounts of cellular protein lysates were separated by SDS‐PAGE electrophoresis and transferred to pure nitrocellulose membrane. After blocking with 5% skim milk, the membrane was probed with different specific primary antibodies overnight at 4°C with one of the following primary antibodies. The following antibodies were used: GAPDH (Sigma), MICAL‐L2 (Thermo Fisher Scientific), EGFR (Cell Signaling, Danvers, MA), HSP27 (Cell Signaling), p‐HSP27 (Cell Signaling), Akt (Cell Signaling), p‐Akt (Cell Signaling), GFP (Cell Signaling), Cdc42 (Cell Signaling), Rac1 (Cell Signaling), vimentin (Cell Signaling), E‐cadherin (BD Transduction Laboratories, Franklin Lakes, NJ), N‐cadherin (BD Transduction Laboratories). After incubation with a secondary antibody for 2 hours at room temperature, the bands were visualized with ECL reagent (Millipore, Billerica, MA). Digital images of the positive bands were detected and analysed with Quantity One (Bio‐Rad, Hercules, CA).

The antibodies for western blotting in our study included anti-β-actin (Abways, Shanghai, China) and anti-CD204 (Abcam, Cambridge, UK).
Western blotting was performed as previously described [59 (link)]. The total protein extraction buffer (Beyotime, Shanghai, China) was used follow the manufacturer's instructions. Further, equal amounts of proteins were separated by SDS-PAGE gel electrophoresis and transferred onto a polyvinylidene fluoride (PVDF) membrane. After blocking with 5% skimmed milk, the membrane was probed with the following primary antibodies: anti-CD204 (1:1000) and anti-β-actin (1:2000). The species-matched secondary antibodies were used (1:10000), and the bands were detected by the Odyssey imaging system (LI-COR, Lincoln, NE, USA).

Cell lysis was performed using a total protein extraction buffer (Beyotime, Shanghai, China) and protein concentration was measured using a BCA Protein Assay Kit (Pierce Biotechnology). Cell lysates were separated by 6–12% SDS-PAGE and transferred to a PVDF membrane. After blocking with 5% skimmed milk, the membrane was probed with one of the following primary antibodies: GRINA (1:1000, ABGENT, AP13558c), β-ACTIN (1:1000, Servicebio, GB13001–3), Bcl-2 (1:1000, Cell Signaling Technology, #4223), Bax (1:1000, Cell Signaling Technology, #5023), Cleaved-caspase3 (1:1000, Cell Signaling Technology, #9664), Cleaved-caspase7 (1:1000, Cell Signaling Technology, #8438), Cleaved-caspase9 (1:1000, Cell Signaling Technology, #9505), Cyclin D1 (1:10000, Abcam, ab134175), Cyclin E (1:1000, Abcam, ab3927), Akt (1:1000, Cell Signaling Technology, #4685), p-Akt (1:2000, Cell Signaling Technology, #4060), P70S6K (1:1000, Cell Signaling Technology, #9202), p-P70S6K (1:1000, Cell Signaling Technology, #9204), 4EBP1 (1:1000, Cell Signaling Technology, #9644), p-4EBP1 (1:1000, Cell Signaling Technology, #2855), AMPK (1:1000, Cell Signaling Technology, #2532), p-AMPK (1:1000, Cell Signaling Technology, #2535), and respective species-specific secondary antibodies (ThermoFisher Scientific). The bound secondary antibodies were detected using the Odyssey imaging system (LI-COR Biosciences, Lincoln, NE).