Anti pi 4 5 p2

The primary antibodies used in this study are as follows: anti-PI(4)P (Echelon Biosciences Inc., Salt Lake City, UT, USA, Z-P004, 10 μg/mL for immunofluorescence (IF), 2.5 μg for immunoprecipitation (IP)), anti-Son (Abcam, Cambridge, UK, ab121759, 1 μg/mL for IF), anti-C23 (Abcam, ab22758, 1 μg/mL for IF), anti-lamin B1 (Abcam, ab16048, 3 μg/mL for IF), anti-PI(4,5)P2 (Echelon Biosciences Inc., Z-A045, 2.5 μg/mL for IF), anti-RPA194 (Santa Cruz Biotechnology, Inc., Dallas, TX, USA, sc-28714, 1 μg/mL for IF), anti-mouse IgM isotype control (Abcam, ab91545, 2.5 μg for IP), anti-hnRNP U (Merck, Darmstadt, Germany, 05-1516, 1 μg/mL for Western blot (WB)), anti-NXF1 (Abcam, ab129160, 0.03 μg/mL for WB) and anti-NUMA1 (Abcam, ab109262, 0.1 μg/mL for WB).
The secondary antibodies used in this study are as follows: goat anti-Mouse IgM Alexa Fluor 555 (Invitrogen, Waltham, MA, USA, A21426, 5 μg/mL), goat anti-Mouse IgM Alexa Fluor 568 (Invitrogen, A21043, 5 μg/mL) and goat anti-Rabbit IgG Alexa Fluor 488 (Invitrogen, A11034, 5 μg/mL) for IF; and IRDye^® 800 CW Donkey anti-Rabbit IgG (LI-COR Biosciences, Lincoln, NE, USA, 926-32213, 0.05 μg/mL) and IRDye^® 800 CW Donkey anti-Mouse IgG (LI-COR Biosciences, 926-32212, 0.05 μg/mL) for WB.

Cell lines or primary neuronal cultures were fixed in cold 2% paraformaldehyde for 20 min, washed in PBS, and permeabilized with 0.5% saponin in blocking solution (1% BSA in PBS (w/v)) for 30 min at room temperature. Cells were incubated with anti-α-Syn antibodies, Syn211 (at 1:750 dilution, overnight at 4 °C); MJFR-1 (1:2000), LB509 (ab27766; 1:250), or anti-α-Syn ab21976 (1:330) from Abcam, Zotal, Tel Aviv, Israel. The immunoreactive pattern of these anti-α-Syn antibodies in SK-Mel2 cells is shown in Fig. S1 (P-AP2M1-T156 (D4F3; Cell Signaling Technology; 1:300). The following antibodies were from Echelon Biosciences (Salt Lake City, UT, USA): anti-PI(3,4)P₂ (Z-P034b; 1:300) or anti-PI(4,5)P₂ (Z-P045 1:200; Z-A045 1:800) for 2 h at room temperature. Cells were then washed (PBS; 10 min ×3) and incubated with a host-suitable secondary ab, washed again, and mounted in Vectashield mounting medium (Vector Laboratory, Burlingame, CA USA).