Enhanced chemiluminescent hrp substrate

Equal amounts of protein were separated by 10% SDS polyacrylamide gel electrophoresis and transferred to PVDF membranes. After blocking with 5% nonfat milk in TBST for 1 h, membranes were incubated overnight at 4°C with Fasn, GAPDH, Wnt5a, Wnt5b, and Fzd2 primary antibodies followed by incubation with HRP‐conjugated secondary antibodies. Immunoreactive bands were detected with enhanced chemiluminescent HRP substrate (Millipore, Bollerica, MA).

To determine protein expression and phosphorylation status, transfected A549 cells with empty vector pcDNA3.1 or pSARS-PLpro were harvested 2 days after transfection. Western blotting of cell lysates was accomplished, as described in our prior reports12 (link)13 (link). Resulting blots were probed with primary antibodies, including mouse polyclonal anti-E. coli synthesized PLpro, rabbit anti-vimentin (GeneTex), rabbit anti-TGF-β1 (Cell signaling), anti-α-SMA (Santa Cruz Biotechnology), rabbit anti-Egr-1, anti-phospho Erk1/2 (Thr202/Tyr204), anti-phospho p38 MAPK (Thr180/Tyr182), anti-phospho STAT3 (Ser727) (Cell Signaling), and anti-β-actin mAb (Abcam). Immune complexes were detected using HRP-conjugated goat anti-mouse or anti-rabbit IgG antibodies, as well as enhanced chemiluminescent HRP substrate (Millipore).

Harvested cells were lysed in ice-cold universal protein extraction buffer (Bioteke, Beijing, China) supplemented with protease inhibitor cocktail (Roche, Basel, Switzerland) for 30 min. Cell lysates were separated on SDS-page gel and transferred to PVDF membrane (Millipore, Billerica, MA, USA). Membranes were blocked in TBS-T Buffer (50 mM Tris-HCl, 150 mM NaCl, 0.1% Tween, PH7.6) supplemented with 5% nonfat dry milk, incubated overnight at 4°C with primary antibodies followed by 5 min washes in TBS-T for three times and incubated with HRP-labeled secondary antibody for 1 hour at RT. Specific proteins were visualized using enhanced chemiluminescent HRP substrate (Millipore). For phosphorylated protein detection, cell lysates were separated on Phos-tag Agarose SDS-PAGE gel (Boppard, Beijing, China) according to the manufacturer's protocol.
For IP assay, cell lysates prepared from 1 × 10⁷ cells were incubated with 0.8μg target antibody for 2 hours at 4°C with gentle inverting (equivalent cell lysates were incubated with IgG as input control), then incubated with 20 μl of protein G&A agarose (Beyotime) overnight, and precipitated by centrifugation at 12 000 g for 1 min. Complexes were washed four times in ice-cold PBS Buffer (pH7.4), and subjected to western blotting.

Equal amounts of protein were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes. Membranes were blocked in TBST containing 5% non-fat dry milk at room temperature for 1h, and incubated with PLD2, GAPDH primary antibodies at 4°C for overnight followed by incubation with horse-radish peroxidase (HRP) conjugated secondary antibodies. Immunoreactive bands were visualized using an enhanced chemiluminescent HRP substrate (Millipore, Billerica, MA, USA).

Cells were harvested with 1x CCLR or RIPA buffer (10 mM Tris buffer at pH 6.5, 150 mM NaCl, 50 mM EDTA, 1% DOC, and 1% NP-40 containing protease inhibitors). The protein concentration of cell lysates was determined with a BCA protein assay kit (23225, Thermo) and iMark microplate absorbance reader (Bio-Rad). Proteins were resolved on a 10% sodium dodecylsulfate (SDS)-polyacrylamide gel for electrophoresis and transferred to polyvinylidene fluoride (PVDF) membranes. Non-fat dry milk (5%) in PBS containing 0.1% Tween-20 (PBST) was used for blocking. After washing three times with PBST, the PVDF membranes were incubated with primary antibodies overnight at 4°C. After primary antibodies were removed, PVDF membranes were washed four times with PBST and incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h at room temperature. Membranes were washed with PBST three times and incubated with an enhanced chemiluminescent HRP substrate (Millipore) for x-ray film detection. The antibodies used in this study were as follows: ARNT (sc-17811, Santa Cruz), α-tubulin (Sigma-Aldrich), and caspase-3 (9665S, Cell Signaling). The expression of protein was confirmed at least for three times.