Atg14

Rabbit monoclonal Abs against p-Akt (#4060), total forms of Akt (#2938), p-mTOR (#5536), mTOR (#2972), p-4EBP1 (#9451), 4E-BP1 (#9452), PAK1 (#2602), HA tag (#3714), Atg14 (#96752), Vps34 (#4263), PI3K (#4249), LC3A/B (#4108), Beclin1 (#3495), and Atg5 (#12994) were purchased from Cell Signaling Technology. Mouse monoclonal Abs against ubiquitin (sc-271289) were purchased from Santa Cruz Biotechnology.
The samples derived from cells and tumor homogenates were lysed in Mammalian Protein Extraction Reagent (Thermo Fisher Scientific), separated by electrophoresis on 12% SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose transfer membranes (Merck Millipore, Cork, Ireland). Proteins were detected using primary Abs at a concentration of 1:1,000 (Cell Signaling Technology) and were incubated overnight. Specific interaction with the primary Abs was detected using corresponding secondary Abs conjugated to HRP (Biosharp), and signals were developed using the enhanced chemiluminescence (ECL) reagents (Biosharp). Gel bands were quantified by ImageJ software, and data are presented as mean ± SD from three independent immunoblotting assays.⁵¹ (link) Phosphorylated and total protein levels were determined and quantified by three successive immunoblotting membranes.

Proteins from kidney lysates or PKD cells were isolated using Radio-Immunoprecipitation Assay (RIPA) Lysis and Extraction Buffer (Thermo Scientific, New York, CA, USA), and protein concentration was measured using Pierce Rapid Gold BCA Protein Assay Kit (Thermo Scientific, New York, CA, USA). Proteins (50 µg) were separated on a 12% SDS-PAGE and transferred to PVDF membranes (Invitrogen, New York, CA, USA). The membranes were blocked with 5% skim milk for 2 h, and incubated with primary antibodies against ULK1 (Invitrogen, USA), LC3-I (Abcam, Cambridge, MA, USA), LC3-II (Abcam, Cambridge, MA, USA), p62 (Cell Signaling Technology, Danvers, MA, USA), E2F-1 (Abcam, Cambridge, MA, USA), PCNA (Cell Signaling Technology, USA), ATG14 (Cell Signaling Technology, Danvers, MA, USA), Beclin1 (Invitrogen, New York, CA, USA) and GAPDH (Cell Signaling Technology, Danvers, MA, USA). Then, the membranes were incubated with HRP-conjugated secondary antibody (Abcam, Cambridge, MA, USA). The bands were visualized using ECL Chemiluminescent Substrate Reagent Kit (Invitrogen, New York, CA, USA). GAPDH was used as an internal control.