Journal: Journal of Biological Chemistry

Article Title: Developmental Control of Apoptosis by the Immunophilin Aryl Hydrocarbon Receptor-interacting Protein (AIP) Involves Mitochondrial Import of the Survivin Protein

doi: 10.1074/jbc.m110.210120

Figure Lengend Snippet: FIGURE 6. Tom20 regulation of mitochondrial import of survivin. A, ali- quots of GST, GST-Tom20, or GST-Tom70 recombinant proteins were mixed with Raji cell extracts, and bead-bound material was analyzed by Western blotting. Bottom panel, Coomassie Blue staining of recombinant GST fusion proteins. B, Raji mitochondrial extract was immunoprecipitated with an anti- body to survivin or IgG, and proteins in pellets or supernatants (unbound) were analyzed by Western blotting. C, recombinant wild-type survivin or the survivin 1–141 mutant and GST-Tom20 beads were mixed with increasing concentration of recombinant AIP, and bead-bound proteins were analyzed by Western blotting (top panel). Bottom panel, GST or GST-Tom20 was incu- bated with recombinant AIP in the presence of increasing concentrations of survivin (up to 0.4 M), and bound proteins were analyzed by Western blot- ting. D, independently established clones (#) of HeLa cells stably transfected with control (Ctrl) or Tom70- or Tom20-directed shRNA were analyzed by Western blotting. E, HeLa cells were transfected with control, non-targeting (Ctrl) or Tom20-directed siRNA and analyzed by Western blotting. TCE, total cell extracts. F, clones of HeLa cells with stable knockdown of Tom70 or Tom20 were treated with 0.5 M staurosporine for 24 h and analyzed for cell viability by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide. DataaremeanS.E.ofreplicates(n3).***,p0.0001.Twodifferentclones per conditions were tested with identical results in two independent experiments.

Article Snippet: Antibodies against AIP (Novus Biologicals), survivin (Novus Biologicals), Tom20 (Santa Cruz Biotechnology), Tom70 (Novus Biologicals), COX-IV (Clontech), Smac (Pro-Sci), -actin (Sigma), and Hsp90 (BD Biosciences) were used.

Techniques: Recombinant, Western Blot, Staining, Immunoprecipitation, Mutagenesis, Concentration Assay, Clone Assay, Stable Transfection, Transfection, Control, shRNA, Knockdown

Journal: Autophagy

Article Title: Deregulated MTOR (mechanistic target of rapamycin kinase) is responsible for autophagy defects exacerbating kidney stone development

doi: 10.1080/15548627.2019.1635382

Figure Lengend Snippet: Antibodies used in this study.

Article Snippet: Antibodies Product SQSTM1/p62 (sequestosome 1) MBL, PM045 phosphorylated SQSTM1/p62 (Ser 351; p-SQSTM1) MBL, PM074 LGALS3/galectin-3 (lectin, galactose binding, soluble 3) Santa Cruz Biotechnology, sc-23,938 mono- and polyubiquitinylated conjugated monoclonal antibody (FK2) BML, PW8810 LAMP1 (lysosomal-associated membrane protein 1) Abcam, ab24170 MAP1LC3 (microtubule-associated protein 1 light chain 3) MBL, PM036 TOMM20 (mitochondrial translocase of outer mitochondrial membrane 20) MBL, ab78547 PINK1 (PTEN induced putative kinase 1) Proteintech, 23,274–1-AP PRKN/PARK2/PARKIN (parkin RBR E3 ubiquitin protein ligase) Cell Signaling Technology, #2132 TFEB (transcription factor EB) Proteintech, 13,372–1-AP RPS6KB1/p70S6K Cell Signaling Technology, #9202 phosphorylated RPS6KB1/p70S6K (p-RPS6KB1/p70S6K) Cell Signaling Technology, #9234 ULK1 (unc-51 like kinase 1) Cell Signaling Technology, #8054 Phosphorylated ULK1 (p-ULK1) Cell Signaling Technology, #14,202 LRP2/MEGALIN (low density lipoprotein receptor-related protein 2) Abcam, ab76969 AQP2 (aquaporin 2) Abcam, ab199975 SLC12A3/thiazide-sensitive NaCl Cotransporter Merck KGaA, AB3553 COX4I1 (cytochrome c oxidase subunit 4I1) Proteintech, 11,242–1-AP 4ʹ,6-diamidino-2-phenylindole (DAPI) Dojindo, 340–07971 ACTB/β-actin Sigma-Aldrich, A5316 histone H3 Abcam, ab1791 Open in a separate window Antibodies used in this study.

Techniques: Binding Assay