Mouse anti p62

Cells plated on coverslips in 12-well plates and fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.1% v/v Triton X-100/1xPBS (10 min), and blocked with 10% normal goat serum prior to incubation with primary antibodies overnight. Rabbit anti-ki67 (Millipore, 1:200), rabbit anti-Tom20 (Thermofisher, 1:1000), mouse anti-cytochrome C (BD, 1:500), rabbit anti-APP (Cell sig, 1:200), mouse anti-P62 (abcam, 1:500) and mouse anti-Tom20 (Santa Cruz, 1:100). Cells were then labeled with appropriate secondary antibody raised in goat (Invitrogen). Images were taken with the LSM800 (Zeiss) confocal microscope by using the 40×/0.5 EC Plan-Neofluar objective or the 63×/1.4 Oil Plan-Apochromat objective. Excitation and acquisition parameters were constrained across all paired comparisons. For fluorescent quantification, morphometric measurements of images were performed using Image-Pro Plus software (MediaCybernetics). Mitochondrial morphology quantification was conducted by the Mito-Morphology Macro^{44 (link)} through ImageJ software (National Institutes of Health) as previous described^{43 (link)}. In brief, images of 30 cells from random view field of each group were first processed with a median filter to obtain isolated and equalized fluorescence, then individual mitochondria were analyzed for the lengths of major axes.

Mature MNs and CNs were fixed with 4% PFA‐PBS for 15 minutes and incubated with 10% goat serum for blocking for 1 hour at room temperature. The cells were incubated overnight at 4°C with rabbit anti‐cleaved‐caspase‐3 (Cell Signaling, 1:350), rabbit anti‐PABP (1:500), mouse anti‐Islet 1 (DSHB, Iowa City, IA, http://www.uiowa.edu/~dshbwww, 1:200), mouse or rabbit anti‐Tuj1 (Covance, Princeton, NJ, http://www.covance.com, 1:1,000), mouse anti‐p62 (Abcam, Cambridge, U.K., http://www.abcam.com, 1:500), rabbit anti‐GA (ProteinTech, 1:500), rabbit anti‐GP (ProteinTech, 1:500), rabbit anti‐PR (ProteinTech, 1:500), rabbit anti‐GR (ProteinTech, 1:500), goat anti‐TIA‐1 (Santa Cruz Biotechnology, Santa Cruz, CA, http://www.scbt.com, 1:500). After washing with 0.1% Triton‐X/PBS three times for 10 minutes, the samples were incubated with Alexa Fluor 488 or Alexa Fluor 568 conjugated goat anti‐rabbit or goat anti‐mouse secondary antibodies (Life Technologies) for 1 hour at room temperature. Nuclei were stained with 4',6‐Diamidino‐2‐Phenylindole (DAPI). Fluorescence was visualized using a confocal microscope Zeiss LSM.

Primary antibodies used were: mouse anti-GFAP (Thermo Fisher Scientific), mouse anti-nestin (Rat-401), mouse anti-p62 (Abcam), rat anti-phosphorylated p62 (MBL: D343-3), mouse anti-ubiquitin (Santa Cruz Biotechnology), rabbit anti-TBK1 (Cell Signaling), rabbit anti- phosphorylated TBK1 (Novus Biologicals), rabbit anti-GFAP (Dako), rabbit anti-Ki67 (Spring Bioscience), rabbit anti-p62 (Enzo), rabbit anti-Sox2 (Millipore), rat anti-Ki67 (BioLegend), and guinea pig anti-doublecortin (anti-DCX; EMD Millipore). Secondary antibodies were goat anti–rabbit IgG-FITC, goat anti–rabbit IgG–Texas red, goat anti–mouse IgG-FITC, goat anti–mouse IgG–Texas red, goat anti–mouse IgG-HRP, and goat anti–rabbit IgG-HRP (Jackson Immunology). Dihydroethidium (DHE) was purchased from Sigma-Aldrich and Amlexanox was purchased from MedChemExpress. Transfections were carried out using Lipofectamine 3000 reagent (Invitrogen).

U2OS WT, ATG7KO, HeLa WT, and HeLa p62KO were seeded into a 12-well plate for 24 h. Cells were then washed twice with PBS and treated for 2 h with EBSS (ThermoFischer, #24010043) and/or 200 nM Bafilomycin A1. For LC3 immunoblotting with rapalog2 treatment, HeLa cells were treated for 4 h with 500 nM rapalog2. Finally, cells were lysed and processed for WB.
Primary antibodies used were: rabbit anti-LC3 (Novus Biologicals, #NB600-1384, 1/1000), mouse anti-p62 (Abcam, #ab56416, 1/1000),mouse anti-actin (Merck, #MAB1501, 1/5000), and mouse anti-tubulin alpha (Sigma, #T5168, 1/20,000). Goat anti-mouse and goat anti-rabbit secondary antibodies conjugated to Alexa Fluor 680/800 were used for visualization and purchased from ThermoFischer Scientific.

For protein extraction, 50 μl of laemmli buffer (0.05% (w/v) bromophenol blue, glycerol (30% (v/v)), 5 M EDTA, NaOH solution, 6% (w/v) SDS and 1.875 M Tris pH 8.8 solution) and 1% (v/v) Dithiothreitol, (all from Sigma-Aldrich, USA) were used. Samples were macerated and denatured for 20 min at 45 °C followed by 20 min 65 °C and finally 10 min at 90 °C. For the immunoblotting assay, 10 μg of total protein extract were resolved in a 12% SDS gel and transferred to a nitrocellulose membrane for 10 min in Trans-Blot Turbo transfer system (Bio-Rad, USA). Membranes were blocked in Tris-buffered saline (TBS) with 0.1% tween 20 (TBS-T) containing 5% BSA and afterwards incubated overnight at 4 °C, with the polyclonal primary antibodies in 1% BSA: rabbit anti-LC3A/B Antibody (1:1000) (Cell-Signaling Technology, USA), mouse anti‐p62 (1:1000) (Abcam, UK), rabbit anti-human caspase 3 (1:700) (Cell Signaling Technology, USA) and mouse anti‐alpha‐actin (Millipore, USA). Secondary antibodies (HRP, anti‐rabbit, anti‐mouse) were from Bio‐Rad, USA (1:5000). Blots were treated with the SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific, USA) or Clarity Western ECL Substrate (Bio‐Rad, USA). Digital images and densitometry analysis of the bands were obtained in a ChemiDoc XRS System (Bio‐Rad, USA) with Quantity One software V4.6.5 (Bio‐Rad, USA), respectively.