Myc tag antibody

Animal tissues were shredded by TissueLyser LT (Qiagen, Hilden, Germany). The shredded tissues and cells were lysed in NP40 buffer (Life Technologies, Carlsbad, USA) containing protease inhibitor cocktail (Roche, Mannheim, Germany). Lysates were mixed with loading buffer and heated at 95 °C for 5 min. Protein samples were resolved on 4–12% SDS–PAGE gels (Life Technologies, Carlsbad, USA) and transferred onto nitrocellulose membranes (GE Healthcare, Freiburg, Germany). Non-specific antibody binding was blocked with 5% nonfat milk in TBST buffer (50 mM Tris/150 mM NaCl/0.1% Tween-20) for 1 h. The membranes were incubated with primary antibodies in incubation solution (2% milk in TBST) overnight. Myc-tag antibody (Cell Signaling #5605, Danvers, USA) was diluted 1:2500; Tubulin antibody (Sigma-Aldrich #T5168, Munich, Germany) was diluted 1:5000; RASAL antibody (Abcam #ab168610, Cambridge, UK; Biorbyt #orb101674, Cambridge, UK) was diluted 1:1000. The membranes were washed three times with 2% milk in TBST and incubated with HRP conjugated secondary antibody for 1 h. Membranes were visualized using LumiGLO chemiluminescence (Cell Signaling, Danvers, USA) and images were documented by a ChemiDoc MP System and processed using ImageLab software (Bio-Rad, Munich, Germany). All uncropped blots are included in Supplementary Fig. 13.

ChIP assay was performed using 1 Day ChIP kit and Shearing ChIP kit (Diagenode, Denville, USA) according to the manufacturer’s protocol. After lentiviral transduction and cross-linking, mouse kidney fibroblasts were fixed with formaldehyde and the DNA was sheared into small fragments. After incubation with a Myc-tag antibody (Cell signaling, Beverly, USA), the pulled-down complexes were de-crosslinked and treated with proteinase K. The purified DNA samples were proceeded further for library preparation by TruSeq RNA Library Prep Kit v2 (Illumina, USA) and quality control and library validation were performed by Fragment Analyzer and Kapa PCR (Illumina, USA), respectively. The fragments were sequenced by an Illumina HiSeq4000 instrument. For data analysis, a previous established protocol^{48 (link)} was followed. Briefly, the raw reads from two independent biological replicates were first concatenated and then peak calling was performed with MACSII in order to obtain the count numbers. We used settings for narrow peaks (200 bp window size, 200 bp gap size, and false discovery rate of 0.01) in all cases.

HEK cells were mock or myc-LC3 DNA transfected for 24 h and then either left in complete media or amino acid starved for 1 h. Alternatively, SMC were transfected with Atg7 and mCherry-Atg3 for 48 h before being left untreated or amino acid starved for 1 h with 100 μm H₂O₂ co-treatment. After incubation cells were rinsed in PBS and then lysed into ice-cold PBS containing 1% Triton X-100 and protease inhibitors (cOmplete, Roche). Cell lysates were then centrifuged at 250,000 r.p.m. for 5 min at 4 °C to pellet insoluble material. Supernatants were added to magnetic beads conjugated to myc-tag antibody (Cell Signaling), with some also added to sample buffer for immunoblot analysis. After 24 h incubation at 4 °C, beads were washed five times with PBS containing 1% Triton-X100. Bound proteins were then eluted by addition of sample buffer and assessed by immunoblot analysis.

Conditionally immortalized podocyte were differentiated per established protocols (PMID: 19955187), harvested, and treated with lysis buffer (Pierce Biotechnology, Rockford, IL) supplemented with phosphatase/protease inhibitor cocktail, 1:100 dilution (Cell Signaling Technology) and 1:100 PMSF (Sigma-Aldrich) for 15 min on ice. Cells were then spun at 14,000 RPM for 10 mins at 4 °C (Eppendorf). Protein Immunoblotting was performed as described previously using a MYC tag antibody 1:000 (Cell Signaling Technology), rabbit polyclonal nephrin antibody1:1000 (Abcam), mouse monoclonal Glepp1 antibody 1:1000 (Santa Cruz), mouse monoclonal WT1 antibody 1:300 (Santa Cruz), rabbit polyclonal CD2AP 1:1000 (Abcam), rabbit polyclonal Synaptopodin antibody 1:800 (Santa Cruz), rabbit polyclonal INF2 antibody 1:1000 (Bethyl Laboratories), and mouse monoclonal β-actin antibody 1:3000 (Sigma-Aldrich)10 (link).

HepG2 cells were used for immunofluorescence experiments. Cells were transfected with TFB1M plasmids (WT and mutants E85A, K86A, D111A, V112A, R183E/R256E/R257E were cloned into p3 × Flag-cmyc vectors) using Lipofectamine 3000. Twenty four hours after the transfection, cells were stained with 500 nM Mitotracker (Invitrogen) for 40 min in an incubator. Cells were fixed in freshly prepared 3.7% paraformaldehyde in PBS for 15 min at RT followed by permeabilization with 0.2% Triton X-100 in PBS for 5 min and blocked with 1% bovine serum albumin in PBS with 0.3% Triton. These cells were incubated with the Myc-tag antibody (Cell Signaling, mouse antibody) in a humidified chamber overnight at 4°C and washed three times with TPBS (0.05% Tween-20 in PBS). Primary antibodies were visualized with FITC-conjugated goat anti-mouse IgG (Jackson ImmunoResearch). DNA was stained with DAPI (Sigma).