Anti rac1 cdc42

Total protein from each group was extracted using radioimmunoprecipitation assay (RIPA) buffer (Thermo Fisher Scientific, United States). After separation via SDS-PAGE, the proteins were transferred onto PVDF membranes. The membranes were incubated overnight at 4°C with the following primary antibodies: anti-cAMP-responsive element-binding protein (CREB) (1:1,000, Cell Signaling Technology Cat# 9197, RRID:AB_331277), anti-p-CREB (1:1,000, Abcam Cat# ab32096, RRID:AB_731734), anti-Rac1/Cdc42 (1:1000, Cell Signaling Technology Cat# 4651, RRID:AB_10612265), anti-p-Rac1/Cdc42 (1:1,000, Cell Signaling Technology Cat# 2461, RRID:AB_2300703), anti-CDH12 (1:10,000), and anti-β-actin (1:2,000), after blocking with 5% skim milk. The membranes were then washed with TBST (TBS with 0.1% Tween 20). After incubation with horseradish peroxidase-conjugated secondary antibodies at 25°C for 2 h, the blot was covered with efficient chemiluminescence solution (Cat# 180e5001, Tanon, Shanghai, China) and visualized using X-ray film. Images were analyzed using ImageJ software (NIH, United States). β-actin was used as the loading control.

Recombinant macrophage colony-stimulating factor (M-CSF) and RANKL and monoclonal anti-histidine antibody were from R&D Systems (Minneapolis, MN). Anti-Rac1/Cdc42, anti-pS71-RAC1/Cdc42, anti-pSer⁴⁷³-Akt, anti-Akt, anti-PAK1/2/3, anti-pSer¹⁴⁴-PAK1, anti-pS21-PAK1, anti-pThr⁴²³-PAK1, anti-PKC substrate serine/threonine motif, and anti-ATM/ATR substrate serine/threonine motif antibodies were products of Cell Signaling Technology (Danvers, MA). Monoclonal antibodies against Flag (M5) and β-actin and anti-M2-conjugated agarose resin were purchased from Sigma (St. Louis, MO). Recombinant human RAC1-GST, Ni-NTA His tag purification kit, and anti-mouse IgG magnetic beads were obtained from Life Technologies (Carlsbad, CA). Rac1 activation assay kit was purchased from Cell Biolabs, (San Diego, CA). Plasmids of pUC57 encoding human Lrrk1 (hLrrk1), mouse Lrrk1 (mLrrk1), ANK truncated mLrrk1 (ΔANK-mLrrk1), LRR truncated mLrrk1 (ΔLRR-mLrrk1), WD40 truncated mLrrk1 (ΔWD-mLrrk1), mLrrk1 kinase domain (mLrrk1-KD), and K651A mutant mLrrk1 (K651A-mLrrk1) were synthesized by GenScript (Piscataway, NJ) and subcloned into the pRRLsin-cPPT-SFFV-GFP-wpre plasmid (Addgene, MA) by replacing GFP with Lrrk1 coding DNA fragments. All constructs contain 3xFlag tags at the NH₂ terminus and 6xhis tags at the COOH terminus.