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3 protocols using sab4200135

1

Generation of RB1CC1 Knockdown Cell Lines

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Stable knockdown cell lines were generated by transfecting PC3 cells with pGIPZ vectors (Open Biosystems) for RB1CC1 (Clone ID #1 V3LHS-307113; Clone ID #2 V3LHS-229368), non-silencing (NS), and GAPDH using Arrest-In Transfection Reagent (Open Biosystems), followed by puromycin selection. These cell lines were named PC3-NS, PC3-GAPDH, PC3-RB1CC#1, and PC3-RB1CC1#2. Expression of RB1CC1 and GAPDH in knockdown cells was analyzed by qPCR. RB1CC1 knockdown was also confirmed by western blot using a polyclonal antibody raised against the c-terminal region (Sigma; SAB4200135). HEK293T and HEK293T transfected with p3XFLAG-CMV10-hFIP200 expression vector (Addgene) were used as controls.
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2

Autophagy Protein Immunodetection Assays

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The following antibodies were used for immunostaining or immunoblotting assays: mouse anti-LC3 (MBL, M152-3), rabbit anti-LC3 (2775S; Cell Signaling Technology), rabbit anti-p62 (MBL, PM045), rabbit anti-FIP200 (SAB4200135; Sigma-Aldrich), rabbit anti-FIP200 (17250-1-AP; Proteintech), rabbit anti-ULK1 (8054; Cell Signaling Technology), rabbit anti-Atg13 (13468S; Cell Signaling Technology), rabbit anti-ATL2 (16688-1-AP; Proteintech), rabbit anti-ATL3 (ab117819; Abcam), mouse anti-WIPI2 (ab105459; Abcam), mouse anti-VAPB (66191-1-IG; Proteintech), rabbit anti-VAPA (15275-1-AP; Proteintech), mouse anti-LAMP1 (553792; BD Biosciences), rabbit anti-PTPIP51 (20641-1-AP; Proteintech), rabbit anti-ATG101 (13492; Cell Signaling Technology), rabbit anti-Myc (2278S; Cell Signaling Technology), mouse anti-Myc (M5546; Sigma-Aldrich), mouse anti-GFP (11814460001; Roche), mouse anti-actin (60008-1-IG; Proteintech), and rabbit anti-HA (H6908; Sigma-Aldrich).
The following reagents were used in this study: wortmannin (PHZ1301; Life Technologies), bafilomycin (B1793; Sigma-Aldrich), rapamycin (R0395; Sigma-Aldrich), Lipofectamine 2000 (12566014; Life Technologies), Lipofectamine RNAi MAX (13778150; Life Technologies), and LysoTracker Deep Red (L12492; Thermo Fisher Scientific).
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3

Antibody Panel for Autophagy Assays

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Primary antibodies used were as follows: rabbit anti‐human Tuj1 (Covance, IF: 1:500), mouse anti‐FLAG (M2, Sigma, WB: 1:2,000, IF: 1:1,000), mouse anti‐Myc (9B11, Cell Signaling, WB: 1:2,000, IF: 1:2,000), mouse anti‐tubulin (DM1A, Sigma, WB: 1:10,000), rabbit anti‐Myc (ab9106, Abcam, WB: 1:2,000, IF: 1:1,000), rabbit anti‐GAPDH (14C10, Cell Signaling, WB: 1:2,000), rabbit anti‐LC3 (2220, Novus Biologicals, WB: 1:1,000), mouse anti‐p62 (610833, BD Biosciences, IF: 1:1,000), rabbit anti‐p62 (18420‐1‐AP, ProteinTech, IF: 1:200), rabbit anti‐FIP200 (SAB4200135, Sigma, WB: 1:500), rabbit anti‐ULK1 (#8054, Cell Signaling, WB: 1:1,000), rabbit anti‐phospho‐ULK S757 (#6888, Cell Signaling, WB: 1:1,000), mouse anti‐EGFP (JL8, Clontech, WB: 1:5,000), rabbit anti‐ATG13 (#6940, Cell Signaling, WB: 1:1,000), rabbit anti‐Rab1a (Ab97956 Abcam, WB: 1:1,000), and rabbit anti‐HA (Sigma, WB: 1:2,000, IF: 1:1,000). Secondary antibodies used for immunoblotting were horseradish peroxidase‐coupled goat anti‐rabbit, goat anti‐rat, and rabbit anti‐mouse IgG (Dako; 1:5,000). Secondary antibodies used for immunofluorescence were Alexa fluorophore (488, 568, or 633)‐coupled goat/donkey anti‐mouse IgG, Alexa fluorophore (488, 568, or 633)‐coupled goat/donkey anti‐rabbit IgG (Invitrogen; 1:500).
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