Mouse monoclonal anti myc

In the co-localization experiments, HEK293 cells were co-transfected with the indicated constructs, using Lipofectamine 2000. After 24 hours, cells were fixed and incubated with 1:500 mouse monoclonal anti-myc (Cell Signaling Technology) and 1:600 rabbit monoclonal anti-FLAG. Slides were imaged by using a C2 confocal microscope (Nikon, Tokyo, Japan), and z-stack images of at least 15 cells per slide were taken.
In autophagy induction experiments, HEK293 cells were transfected with a pEGFP-LC3 plasmid (Addgene #24920) and the indicated construct, using Lipofectamine 2000. After 24 hours, cells were starved for 18 hours in Hank’s Balanced Salt solution supplemented with 0.1 μmol/L of Bafilomycin A1 (Sigma). Cells were imaged as described above and autophagosomes were counted by using NIS-Elements Advanced Research software (Nikon).

Mouse monoclonal anti-β-actin (Sigma, #A1978, 1:5000 for WB); mouse monoclonal anti-myc (Cell Signaling Technology, #2276, 1:4000 for WB; 1:1000 for IF), goat polyclonal anti-VDAC (SantaCruz Biotechnology, #sc-8828, 1:1000 for WB), rabbit polyclonal histone H3 (AbCam, #ab1791), rabbit polyclonal cadherin antibody (NeoBioLab, #A1550, 1:3000 for WB), mouse monoclonal striatin3 antibody (S68) (Pierce, #MA1–46461, 1:3000 for WB; 1:5000 for IF and Santa Cruz Biotechnology, #sc-13562), phospho-GSK-3β (Ser9) antibody (CST, #9336), P-Thr-polyclonal rabbit antibody (CST, #9381), mouse monoclonal P-Ser antibody (Sigma,#P3430), mouse monoclonal anti-cyclinD1 (SantaCruz #sc20044).

The following primary antibodies were used: Rabbit polyclonal anti-SNX18 IgG (AbCam, ab99035); mouse monoclonal anti-myc (Cell Signaling Technology, 2272); Rabbit polyclonal anti-β-Tubulin (LI-COR Biosciences, 926-42211); mouse monoclonal anti-α-tubulin Clone DM 1A (Sigma Aldrich, T9026), anti-α1 Na+/K+ ATPase antibody (ab7671, Abcam), mouse monoclonal anti-LPS (Abcam, ab 8274); Mouse anti-Phospho-Tyrosine antibody, clone 4G10 (Millipore, 05-1050); mouse monoclonal anti-HA (Covance); Rabbit monoclonal anti-Akt (pan) C67E7 (Cell Signaling, 4691); and anti-phospho-Akt (Ser473) D9E (Cell Signaling, 4060). Goat anti-mouse coupled to Alexa Fluor 405, 546 or 647 (Invitrogen) were used as secondary antibodies for immunofluorescence. The IRDye800CW goat anti-mouse IgG; IRDye800CW donkey anti-rabbit IgG; IRDye680LT goat anti-rabbit IgG; and IRDye680LT donkey anti-mouse IgG were purchased from LI-COR Biosciences. Phalloidin-Alexa Fluor 635, fixable analog of lipophilic membrane stain FM 4-64FX and Wheat germ agglutinin (WGA) coupled to Alexa Fluor 647 were from Invitrogen (A34054, F34653, and W324666). Selective inhibitor of Rac1-GEF interaction NSC 23766 was purchased from Tocris Bioscience and the Akt1/2 kinase inhibitor from Sigma Aldrich.

Polyclonal anti-Ush2a (cytoplasmic tail) antibodies that were used for Western blot analysis (WB; 1:1000) and immunostainings (IF; 1:200) were previously validated [57 (link)]. Commercially available antibodies were used, as follows: monoclonal anti-HA (rat) from Roche Diagnostics (Mannheim, Germany; WB 1:500; IF 1:50), anti-dsRed2 (WB 1:2000) Chromotec (Martinsried, Germany), mouse monoclonal anti-Myc from Cell Signaling (Danvers, MD, USA), monoclonal anti-acetylated tubulin from Sigma (Deisenhofen, Germany; IF 1:4000), and goat polyclonal anti-Pericentrin 2 (1:200) Santa Cruz Biotechnology (Heidelberg, Germany). For Western blotting, the secondary antibodies goat-anti-rabbit, goat-anti-mouse, goat-anti-rat; conjugated to Alexa680; (WB 1: 20,000) obtained from Molecular Probes (Darmstadt, Germany) were used. For immunocytochemistry, goat-anti-rabbit, donkey-anti-goat, goat-anti-mouse antibodies conjugated to Alexa488 or Alexa647 (IF 1:400), Molecular Probes (Darmstadt, Germany), respectively, were used and nuclear DNA was stained with DAPI (4’, 6-diamidino-2-phenylindole) (1 mg/mL) (Sigma–Aldrich, Germany).

All plasmids were cloned and amplified using DH5α Competent Cells (Invitrogen, Cat. No. 18265–017). The plasmids encoding EGFP-SKL, myc-ACBD5(human), FLAG-ACBD5(human)-FFAT, ACBD5(human)-ACB and FLAG-ACBD5(human)-WT were generated as described in a previous publication (16). The plasmids mCherry2-C1, mPlum-N1, mPlum-Mito-3 and mCherry-Mito-7 were gifts from Michael Davidson (Addgene plasmid #54563, #54629, #55988, #55102) [20 ]. The primary and secondary antibodies used in this study include mouse monoclonal anti-GFP (MAB3580, Chemicon), mouse monoclonal anti-myc (#2276, Cell signaling), rat monoclonal anti-RFP (5F8, Chromotek), rabbit polyclonal Pex14 (kind gift from D. Crane, Brisbane, Australia), rabbit polyclonal ACBD5(human) (HPA012145, Sigma), rabbit polyclonal VAPB (HPA013144, Sigma), Alexa Fluor 488 anti-mouse IgG, Alexa Fluor 568 anti-rat IgG and Alexa Fluor 647 anti-rabbit (A32723, A11036, A32733, Invitrogen).