Anti pp1 antibody

The protocol was previously described[18 (link)]. Briefly, cells (5x10⁶) were lysed for 20 min at 4°C in lysis buffer (50 mM Tris pH8, 1% NP40, 137 mM NaCl, 1 mM MgCl₂, 1 mM CaCl₂, 10% glycerol and protease inhibitor mixture Sigma Aldrich). Lysates (500 μg) were immunoprecipitated with the appropriated antibody overnight at 4°C and protein A/G Sepharose (Santa Cruz) was added for 1h at 4°C. After washing with 1x TBST (20 mM Tris-HCl pH7.5, 150 mM NaCl, 0.05% Tween 20), the PP1/LRRK2 interaction was competed using 1 mM of the Mut3DPT-LRRK2-Long or Mut3DPT-LRRK2-Shor peptide for 30 min at room temperature. After several washing steps, immunoprecipitates were separated by SDS-PAGE, transferred to nitrocellulose and blotted with anti PP1 antibody (Santa Cruz or Thermo Fisher 1:500 dilution). The membrane was washed and incubated with PO-conjugates secondary antibody (Dako, 1:1000 dilution). Protein detection was performed using the ECL system (Bio-Rad). As internal control, the blot was also hybridized with anti-LRRK2 antibody (Abcam, ab133474, 1;500 dilution). Western blots were densitometred using Image J. Statistic analysis were done using Anova.

Human or mouse fibroblasts were incubated in Cell lysis buffer (P0013J, Beyotime, China) with protease inhibitor (14001, Bimake) at 4 °C for 15 min and then centrifuged at 12,000 × g for 15 min. The lysates were then incubated with anti-human PEAR1 monoclonal antibody LF2 and LF3, anti-mouse PEAR1 monoclonal antibody LF1, anti-PP1 antibody (SC7482, Santa Cruze) overnight at 4 °C, respectively. Mouse IgG (5415, Cell Signaling Technology) was used as a negative control. On the next day, protein A/G agarose beads (SC2003, Santa Cruze) were added to the lysates and incubated for 3 h. Then the agarose beads were harvested and washed 3 times with PBS by centrifugation at 12,000 × g for 1 min, followed by boiling for 5 min at 100 °C in Samples were analyzed by Western blotting with the indicated antibodies.

Cell treated as indicated were harvested. The protein concentration was analyzed by BCA protein Assay Reagent (Sangon Biotech, Shanghai, China). Soluble lysates containing about 20 μg proteins per sample were resolved with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto polyvinylidene fluoride membranes. After blocking with 5% BSA, membranes were incubated with primary antibodies at 4°C overnight and secondary antibodies at room temperature for 1 h. The membrane signals were detected using an Enhanced Chemiluminescent Western Blotting Detection System (Millipore, Billerica, MA, USA) in accordance with the manufacturer's instruction. Antibodies against ROCK, PTEN, p-PI3K, PI3K, p-Akt, Akt, cofilin-1, p-cofilin-1, and GAPDH were from Cell Signaling Technology (Danvers, MA, USA). Antibodies against cleaved ROCK were purchased from Abcam (Cambridge, MA, USA). An anti-PP1 antibody was obtained from Santa Cruz Biotechnology (Dallas, TX, USA).