Western and ip lysis buffer

Western blot assay was conducted as previously described [23 (link)]. In brief, the tissue samples and cells were homogenized and lysed in RIPA Lysis Buffer (P0013B, Beyotime) and Western and IP Lysis Buffer (P0013, Beyotime) with 1 mM phenylmethylsulfonyl fluoride (PMSF). The cell lysate was centrifuged at 4 °C for 15 min, and the cell supernatant was collected. The concentration of extracted proteins was measured using the bicinchoninic acid (BCA) Protein Assay Kit (P0012, Beyotime). A total of 40–80 µg proteins were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by transfer to 0.22 µm polyvinylidene difluoride (PVDF) membranes (Merck Millipore, Burlington, MA, USA). The membranes were incubated with 5% fat-free milk for 1 h at room temperature (RT), followed by incubation with the primary antibodies (1:1000) overnight at 4 °C and appropriate horseradish peroxidase-linked secondary antibodies (1:5000) for 1 h at RT. Immunoreactive species were visualized using enhanced chemiluminescence (ECL) detection Kit (ORT2655, PerkinElmer, Waltham, MA, USA) and GE Amersham Imager 600 (GE, Chicago, IL, USA). β-actin was used as a loading control to normalize protein loading. The intensities of immunoreactive bands were analyzed using ImageJ 1.52a software from the National Institutes of Health (Bethesda, MD, USA).

Protein carbonyl content [61 (link)], MDA content [62 (link)], and antioxidant enzyme activities [62 (link)] were determined as described previously. Briefly, synchronized L1 nematodes were pretreated with 0.5 mg/mL of nostoglycan and then exposed to 10 mM paraquat as above. Approximately 3000 nematodes were homogenized in 400 μL of Western and IP lysis buffer (Beyotime, Shanghai, China). The lysate was collected by centrifugation and used for the following determinations using commercial assay kits, respectively: protein carbonyl content (Jiancheng, Nanjing, China); MDA content and SOD, CAT and GPx activities (Beyotime); and protein content (Thermo), which was used to normalize protein carbonyl and MDA contents and antioxidant enzyme activities.

Tissue and cellular samples were washed with ice-cold phosphate-buffered saline (PBS) and lysed with Western and IP lysis buffer (Beyotime, Jiangsu, China). The lysates were homogenized and centrifuged at 13,000 g for 15 min at 4 °C. Then, the protein concentrations of the collected supernatants were determined using a BCA Protein Assay kit (Pierce, Rockford, IL, USA). The extracted protein samples (15–20 μg per lane) were subjected to 8%, 10% or 12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and the separated proteins were transferred to nitrocellulose membranes (PerkinElmer Life Science, Boston, MA, USA) and blocked with 5% nonfat milk/TBST for 1-2 h at room temperature. The membranes were incubated with primary antibodies at 4 °C overnight, after which they were incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. Western blots were developed using an ECL Western blotting detection kit (Pierce, Rockford, IL, USA) and quantified by scanning densitometry.

To directly detect K48 ubiquitinated and total ubiquitinated YAP enriched in cell extracts, HA-K48 ubiquitinated or HA-Ub plasmids, Myc-YAP and Flag-RNF31 or Flag-Vectors were transfected into HEK293T cells. After 24 hours, 10 μM MG132 was added to the cells for treating 8 hours. Then, cells were lysed with Western and IP lysis buffer (P0013J, Beyotime) with a protease inhibitor cocktail (Roche). Total cell lysates were incubated with 30 μl of Protein Agarose (Beyotime, P2012) for preclearing 2 h at 4 °C, and immunoprecipitation was then performed with anti-YAP antibody (14,074, Cell Signaling Technology) or Rabbit IgG (Beyotime, A7016) for 4 h at 4 °C. Western blot was performed with anti-HA antibody to detect YAP total polyubiquitinated or YAP K48 polyubiquitinated.

Fragments of bbYY1 or hsYY1 were inserted into the pGEX-6P-1 vector for GST-fused protein constructions. The constructs were then transformed into E. coli. BL21 bacteria. The transformed bacteria were cultured at 37 °C to an OD = 0.6–0.8, chilled to 18 °C and induced with 0.5 mM IPTG. After 12 h of induction, the cells were collected, resuspended, and sonicated with lysis buffer (TBST with protease inhibitor cocktail). The lysate supernatant was incubated with 40 μL of Glutathione-Sepharose slurry (cat.: 17-5113-01, GE) for 1 h at 4 °C to pull down GST-fused protein.
The MBP-bbRAG1L/2L expression vectors were co-transfected into 293 T cells for MBP fusion protein preparation. The transfected cells were continually cultured 60 h after vector transfection and lysed using western and IP lysis buffer (cat.: P0013, Beyotime Biotechnology). The lysate supernatant was rotated with 40 μL of the above well-prepared slurry (containing GST or GST fusion protein) in TBST buffer (with protease inhibitor cocktail addition) at 4 °C overnight and washed four times with TBST. Finally, the slurry binding with proteins was eluted in Laemmli buffer (supplemented with 200 mM 2-mercaptoethanol) and resolved on 10% SDS-PAGE gels for further western blotting analysis. The ECL chemiluminescence method was used for the western blot analysis.

LMH cells were washed with PBS (pH 7.0) and lysed with Western and IP lysis buffer (Beyotime Biotechnology) at 4°C for 30 min. Cell lysates were incubated for 10 h with Fc-tagged fibre 1 or Fc protein (as a negative control) and protein A/G agarose antibody. Precipitates were washed five times in Western and IP lysis buffer and once in PBS, and subsequently centrifuged 5000 rpm for 5 min (Eppendorf). The proteins binding in agarose were separated by SDS-PAGE and visualized by silver staining (Thermo Fisher Scientific) according to the manufacturer’s instructions. Differential bands between lysate incubated with fibre 1 and negative control lysate were excised from the gel and transferred to Shanghai Hoogen Biotechnology Company for mass spectrometry (MS) analysis.

After various treatments, cells were washed twice with ice-cold PBS and lysed with Western and IP lysis buffer (Beyotime, Haimen, China) containing 1 mM phenylmethanesulfonylfluoride on ice. The lysates were centrifuged at 13,000× g for 6 min at 4 °C. Protein concentrations of the collected supernatants were quantified with BCA kit (Lot No.: 23225, Pierce Biotechnology, Rockford, IL, USA). Equal aliquots of protein samples were subjected to SDS-PAGE, and then transferred to NC membranes. After blocking with 5% (w/v) skimmed milk for 1 h at room temperature, membranes were incubated with primary antibodies (1:1000 dilution) at 4 °C overnight. After washing with TBST three times (15 min each), the membranes were incubated with horseradish-peroxidase-conjugated secondary antibodies (1:4000 dilution) for 1 h at room temperature. After washing with TBST three times, chemiluminescent detection was performed using ECL western blotting substrate (Bio-Rad, Hercules, CA, USA) and quantified by scanning densitometry.