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2 protocols using rabbit anti vps34

1

Comprehensive Antibody Panel for Cellular Analysis

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The following primary antibodies were employed for either immunofluorescence or western blotting: mouse anti-ARH-I (for IF 1:250, for WB 1:1000; cod. ab45768; Abcam, Cambridge, UK), mouse anti-p21 (for IF 1:100, for WB 1:200; cod. sc-817; Santa Cruz Biotechnology, Dallas, TX, USA), rabbit anti-LC3 (1:2000; cod. L7543; Sigma-Aldrich), rabbit anti-p62 (1:500; cod. 8025; Cell Signaling, Danvers, MA, USA), mouse anti-β-actin (1:2000; cod. A5441; Sigma-Aldrich), mouse anti-BECLIN-1 (1:500; cod. 612112; BD Biosciences, Franklin Lakes, NJ, USA), goat anti-BECLIN-1 (1:250; cod. sc-10086; Santa Cruz Biotechnology), rabbit anti-VPS34 (1:500; cod. 4263; Cell signaling), mouse anti-STAT3 (1:500; cod. 9139; Cell Signaling), rabbit anti-phospho (Tyr705) STAT3 (1:1000; cod. 9145; Cell Signaling), rabbit anti-Ki-67 (1:100; cod. HPA001164; Sigma-Aldrich), mouse anti-BCL-2 (1:500; cod. 15071; Cell Signaling), mouse anti-IL-6R (1:1000; cod. AHR0061; Invitrogen, Waltham, MA, USA), rabbit anti-cyclin D1 (1:500; cod. 2978; Cell Signaling), rabbit anti-p38 (1:500; cod. 9212; Cell Signaling), rabbit anti-GAPDH (1:1000, cod. G9545, Sigma Aldrich), and mouse anti-ERK1/2 (1:500; cod. 05-1152; Millipore, Burlington, MA, USA).
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2

Molecular Cloning and Tagging Strategies

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Human Dpr1 cDNA was cloned into pEGFP-C3 to generate GFP-Dpr1. Human Vps34 cDNA was cloned into the KpnI-XbaI sites of the pcDNA3.1(+)-HA or pCS2(+)-Flag vector. The Flag-Beclin1 was a gift from Dr Honggang Wang. The p62-GFP was a gift from Dr Terje Johansen. Dvl2 was cloned into the Bam HI site of pDsRed-N1. Atg14L was cloned from Atg14L-GFP into EcoRI-SalI sites of pmCherry-N1. Dpr1 shRNAs were purchased from Open Biosystems. MG132 was from Calbiochem and prepared as 100 mg/ml in DMSO. BafA1, rapamycin, and XAV939 were obtained from Sigma. A769662 was from Abcam. Antibodies include: mouse anti-Calbindin (Sigma), rabbit anti-GFAP (Bioss, Beijing), mouse anti-ubiquitin (FK2 MBL), mouse anti-Beclin1 (BD Biosciences), rabbit anti-Beclin1 (MBL), rabbit anti-Vps34 (Cell Signaling), rabbit anti-Atg14L (MBL), mouse anti-Flag M2 (Sigma), rabbit anti-Dvl2 (Cell Signaling), mouse anti-tubulin and anti-GFP (Santa Cruz Biotech), mouse anti-p62 and rabbit anti-LC3 (MBL).
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