Ab23666

The following primary antibodies were used for immunofluorescence (IF) or immunoblotting (IB): rabbit anti-ATG3 (Abcam, Cambridge, UK; ab108251; IF, 1:50; IB, 1:1000), anti-ATG4B (CST, Leiden, The Netherlands; 5299; IF, 1:100; IB, 1:1000), anti-ATG7 (Abcam, ab133528; IB, 1:1000), anti-SQSTM1 (Abcam, ab109012; IF, 1:200; IB, 1:20,000), anti-OPTN (Abcam, ab23666; IF, 1:50; IB, 1:1000), anti-CACO2/NDP52 (Abcam, ab68588; IF, 1:100; IB, 1:2000), anti-LC3B (CST, 2775; IB, 1:1000), anti-TUBA1A (Santa Cruz Biotechnology, Heidelberg, Germany; SC-5546; IB, 1:1000), anti-USP30 (Abbkine Scientific, Wuhan, China; ABP52679; IB, 1:1000), anti-DNM1L (Cusabio, Houston, TX, USA; CSB-PA002203; IB, 1:1000), mouse anti-PEX14 (IF, 1:100) [40 (link)], and anti-actin (Sigma, A5316; IB, 1:10,000). The secondary antibodies for IF were conjugated to Alexa Fluor 488 (Invitrogen, A11017; 1:2000) or Texas Red (Calbiochem, Darmstadt, Germany; 401355; 1:200), and the secondary antibodies for IB were conjugated to alkaline phosphatase (Sigma, A3687 and A2429; 1:5000 and 1:10,000, respectively).

Neurons were fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.3% Triton-X-100/PBS for 10 min and blocked for 1 h at room temperature (1% BSA/PBS). Neurons were incubated with primary antibodies diluted in blocking buffer at 4 °C for 1 h or overnight. The following primary antibodies were used: mouse anti-SYNJ2BP (1:50 dilution; Sigma-Aldrich, SAB1400613), rabbit anti-phospho-ubiquitin S65 (1:200 dilution; Millipore, ABS1513-I), rabbit anti-optineurin (1:500 dilution; Abcam, ab23666) and mouse anti-βIII tubulin (2G10) (1:1,000 dilution; Invitrogen, MA1-118). Neurons were incubated with secondary fluorescent antibodies (goat anti-mouse IgG (H + L) Alexa Fluor 568 (1:500 dilution; Invitrogen, A11004) and goat anti-rabbit IgG (H + L) Alexa Fluor Plus 647 (1:500 dilution; Invitrogen, A32733)) diluted in blocking buffer for 2 h at room temperature. The coverslips were mounted in Fluoromount G (Invitrogen) and imaged at an Eclipse Ti2 spinning-disk microscope (Nikon) using a ×60 NA 1.40 oil-immersion objective.

Primary antibodies for rabbit anti-OPTN (ab23666) and rabbit anti-beclin-1 (ab62557) were purchased from abcam (Cambridge, MA, USA); mouse anti-LC3 was purchased from MBL International Corporation (M152-3, Woburn, MA, USA); mouse anti-alpha-synuclein was purchased from BD Transduction Labs (610786, San Jose, CA, USA); chicken anti-MAP2 (AB5543), sheep anti-tyrosine hydroxylase (AB1542), anti-tryptophan hydroxylase (AB1541), and anti-choline acetyltransferase (AB1582) were purchased from EMD Millipore (Billerica, MA, USA). Normal donkey serum (017-000-121) and secondary antibodies, Alexa Fluor^®; 647 anti-sheep (713-605-147), Alexa Fluor^®; 488 anti-rabbit (711-545-152), and Cy^TM3 anti-mouse (715-165-151) were purchased from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA, USA). Rotenone (R8875) and glycerol (G5516) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Triton X-100 was purchased from Fisher BioReagents (BP151-500, Fair Lawn, NJ).