Rabbit anti phospho erm

Chicken anti-GFP (Abcam) was used to detect pcms-EGFP co-transfected in primary neurons. Mouse (clone M2) and rabbit anti-Flag and anti-V5 antibodies were from Sigma. Rabbit anti-phospho-ERM (Cell Signaling Technology; MA, USA) was used in an ELISA to detect phosphorylated LRRKtide. Rabbit monoclonal anti-LRRK2 (clone #c41-2; and clone UDD3; Epitomics/Abcam) was used to label LRRK2 by Western immunoblot. For the detection of phosphorylated LRRK2 we employed: rabbit monoclonal anti-pS935, (from Epitomics/Abcam), and rabbit polyclonal anti-pS1292 (generously provided by Genentech; [11 (link)]). For the detection of activated caspase-3, fixed cells were immunostained with active caspase-3 (R&D Systems; MN, USA).

Primary cultured astrocytes grown on coverslips were incubated with recombinant mouse OPN (1 mg/mL) or recombinant human TNF-α (50 ng/mL: Pepro Tech) for 6 h at 37 °C under a 5% CO₂/95% air atmosphere. To detect actin, the cells were washed with PBS, fixed in 4% PFA/0.1 M PB (pH 7.4) for 15 min at room temperature, and treated for 15 min with PBS containing 0.2% Triton X-100. G- and F-actin were then labeled using Alexa488-conjugated deoxyribonuclease I (1:500, Molecular Probes) and rohdamine-conjugated phalloidin (1:1,000, Invitrogen), respectively. For phospho-ERM labeling, the cells were washed with PBS containing 30 mM glycine (G-PBS), fixed with ice-cold 10% trichloroacetic acid for 15 min, and treated for 15 min with G-PBS containing 0.2% Triton X-100. After blocking the cells for 1 h in G-PBS containing 2% BSA and 3% normal goat serum, they were incubated for an additional 1 h with rabbit anti-phospho-ERM (1:400, Cell Signaling Technology). The immune-signals were then visualized using Alexa Fluor568-labeled goat anti-rabbit IgG (1:400, Molecular Probe), and nuclei were visualized using DAPI (1:1,000, Molecular Probe). For Western blot analyses, cell lysates were prepared in RIPA buffer containing 1x protease inhibitor cocktail as described above, and were probed with rabbit anti-ERM (1:1,000, Cell Signaling Technology) or anti-phospho-ERM.

The cells were fixed with 4% paraformaldehyde (PFA), permeabilized with 0.5% Triton X-100/PBS and blocked with 10% FBS/PBS. The primary antibodies used were: mouse mc anti-MAP4K4 (MO-7, clone 4A5, Abnova 1∶100), rabbit anti-ERM (3142, Cell Signalling, 1∶100), rabbit anti-Phospho-ERM (3149, Cell Signalling, 1∶100), mouse anti-p53 (2524, Cell Signalling, 1∶100). The secondary antibodies used in immunostaining were: Cy3-conjugated Donkey Anti-Rabbit IgG (H+L) (711-165-152, Jackson Immunoresearch, 1∶500), Cy5-conjugated Rat Anti-Mouse IgG (H+L) (415-175-166, Jackson Immunoresearch, 1∶500), Phalloidin, Tetramethylrhodamine B isothiocyante (P1951, SIGMA-ALDRICH, 1∶1000), Texas Red-X phalloidin (T7471, Molecular Probes, 1∶1000), Alexa Fluor 488 anti-mouse IgG, (A11029, Invitrogen, 1∶1000), Alexa Fluor 488 anti-rabbit IgG (A11034, Invitrogen, 1∶1000), Texas Red-X anti-mouse IgG (T862, Invitrogen, 1∶1000), Texas Red-X anti-rabbit IgG (T6391, Invitrogen, 1∶1000), phalloidin-488 (Molecular Probes, 1∶500)