Ripa strong lysis buffer

Cells (3.0 × 10⁷) were treated with 0.5 mM NAC, BA (12.5, 25, 50 or 100 μg/ml) and an equal volume of RPMI-1640 medium for 2 h. HPS (3.0 × 10⁷ CFU/ml) was added and the protein was extracted after 6 h using RIPA Lysis Buffer Strong (P0013, Beyotime Biotechnology, Shanghai, China). The protein concentration was determined by BCA protein assay kit (P0012, Beyotime Biotechnology). The proteins were separated by 10% SDS-PAGE and transferred onto a PVDF membrane (60 mA, 1 h). The PVDF membrane was blocked with 5% skimmed milk at 20 °C for 3 h, then incubated with the primary antibody (containing 5% BSA TBS-T solution, 1:1000 dilution, Anti β-actin, cleaved caspase-3, JNK, p-JNK, p38, p-p38, ERK, p-ERK, PKC-α and p-PKC-δ, Cell Signaling Technology, MA, USA; and p-PKC-α and PKC-δ, Sigma, USA) overnight at 4 °C and the secondary antibody (containing 5% skimmed milk TBS-T solution, 1:2000 dilution; Cell Signaling Technology) at room temperature for 3 h. Afterwards, the membrane was visualized by ECL solution (Thermo Pierce ECL, USA) using a ChemiDoc MP Imaging System (Bio-Rad, Hercules, CA, USA). The β-actin was used as an inner loading control. Gray value was analyzed and the relative expression level of protein was obtained.

Map2k5^+/– mutant mice and WT littermates (female, aged 21 weeks, n = 4 per group) were perfused with PBS and sacrificed. Brains were then dissected quickly within 10 min and snap frozen in liquid nitrogen. Proteins were prepared from the ventral midbrain (Yun et al., 2018 (link)) including nigrostriatal dopaminergic neurons. Cytoplasmic proteins were extracted by RIPA Lysis Buffer (Strong) (Beyotime Biotechnology, Shanghai, China) supplemented with Complete Protease Inhibitor Cocktail (10×) and PhosSTOP^TM Cocktail (10×) (Roche Ltd., Basel, Switzerland). Western blots were performed using sodium dodecyl sulfate polyacrylamide gel electrophoresis and nitrocellulose membranes. Primary antibodies included anti-tyrosine hydroxylase Ab (1:1,000, MAB318, Millipore Co., Massachusetts, United States), Erk5 Antibody (1:1,000, #3372, CST Ltd., Boston, United States), Phospho-Erk5 (1:1,000, Thr218/Tyr220), Antibody (1:1,000, #3371, CST), MAP2K5 (Concordet and Haeussler, 2018 (link)). Antibody (1:200, sc-135986, Santa Cruz Biotechnology, Inc., Northern California, United States), and GAPDH (D16H11) XP^® Rabbit mAb (1:1,000, #5174, CST). Signals were detected with an ECL agent (Millipore). Blotting images were acquired and quantitatively analyzed by Image Lab Software (Bio-Rad, CA, United States).

Cell samples were collected and quickly incubated with cold RIPA‐strong lysis buffer (Beyotime Biotechnology) which contains 1% protease inhibitor cocktail (Cwbiotech) and 1% phosphatase inhibitor (Cwbiotech) over 30 min. The lysis sample was then centrifuged at 12,500 rpm at 4°C for 30 min. The supernatant was collected, and a BCA (Thermo Fisher Scientific) assay was used for protein quantitation. A total of 30 μg of protein sample was added to each lane of a sodium dodecyl sulphate–polyacrylamide gel. Proteins were transferred to a polyvinylidene fluoride membrane (0.2 μm; Millipore) after electrophoresis. The membrane was then blocked with 5% skimmed milk at room temperature for 1 h and incubated with the indicated primary antibodies overnight at 4°C. All primary antibodies used in this research were diluted in 1:1000. Later, the membranes were washed with PBST buffer and incubated with secondary antibody for 2 h at room temperature. The secondary antibodies were diluted in 1:10,000. Finally, the targeted protein band was illuminated with ECL Western Blotting Substrate (Millipore).

Cells were lysed with RIPA strong lysis buffer (Beyotime, China) supplemented with the protease inhibitors, 1% PMSF (Roche, Mannheim, Germany), and PIC (Roche, Switzerland). After centrifugation for 15 min, protein concentration in the supernatant was measured using a Pierce™ BCA protein assay kit (Thermo). The supernatant was then mixed with protein loading buffer (NCM Biotech) and boiled at 100 ℃ for 5 min. The same amounts of proteins (30 μg) from all samples were separated by 10% SDS-PAGE gel (Bio-Rad, USA) and transferred onto a PVDF membrane (Immobilon®-P). After blocking in 5% BSA for 1 h at room temperature and incubated overnight at 4 °C with the indicated primary antibodies. Followed by incubation with secondary antibodies for 2 h at room temperature, and visualized by the NcmECL Ultra (A+B) (NCM Biotech) chemiluminescence reagent, GAPDH was used as an internal control. Antibodies against CDK19 were purchased from Proteintech (13761–1-AP, 1:1000) and GAPDH was purchased from Utibody (UM4002, 1:2000). Protein bands were analyzed using Image J software.