plasmid encoding human bag6  (Addgene inc)

Journal: Cell Reports

Article Title: Engineering of immune checkpoints B7-H3 and CD155 enhances immune compatibility of MHC-I −/− iPSCs for β cell replacement

doi: 10.1016/j.celrep.2022.111423

Figure Lengend Snippet:

Article Snippet: Human/Mouse/Rat BAT3/BAG6 antibody , Biotechne , Cat#AF6438; RRID: AB_10717417.

Techniques: Recombinant, Staining, Cell Isolation, Plasmid Preparation, Software

Journal: Cell reports

Article Title: Molecular determinants of the crosstalk between endosomal microautophagy and chaperone-mediated autophagy

doi: 10.1016/j.celrep.2023.113529

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: The dilutions and sources of antibodies used for immunoblot (IB), immunoprecipitation (IP), cell immunofluorescence (cell-IF) and isolated LE/MVB immunofluorescence (LE-IF) in this study were as follows: (dilutions, commercial source, and catalog number indicated in parentheses): rabbit anti-aldolase (1:1000 (IB), Cell Signaling, 3188S), rabbit anti-Alix (1:500 (IB), Novus, NBP1–90201), mouse anti-Alix (3A9) (1:100 (LE-IF), Cell Signaling, 2171S), mouse anti-α synuclein (211) (1:2000 (IB), Santa Cruz, sc-12767), rabbit anti-ATP6V1B (1:1000 (IB), Abcam, ab200839), rabbit anti-Bag1 (1:1000 (IB), Abcam, ab32109), rabbit anti-Bag3 (1:1000 (IB), Novus, NBP2–27398SS), sheep anti-Bag6 (1:200 (IB) and 1:100 (LE-IF), R&D Systems, AF6438), rabbit anti-Bag6 (1:1000 (IB), 1:100 (LE-IF), 1μg (IP), Abcam, ab137076). rabbit anti-Cathepsin B (1:1000 (IB), Abcam, ab214428), mouse anti-cathepsin D (D-7) (1:500 (IB), Santa Cruz, sc-377299), rabbit anti-cathepsin L (H-80) (1:1000 (IB), Santa Cruz, sc-10778), rabbit anti-CHIP (C3B6) (1:1000 (IB), Cell Signaling, 2080), rabbit anti-Cyclophilin A (1:1000 (IB), Cell Signaling, 2175), mouse anti-EEA1 (1:1000 (IB), BD Biosciences, 610456, RRID AB_397830), rabbit anti-GAPDH (1:2000 (IB), Cell Signaling, 2118S), rabbit anti-GBA (1:1000 (IB), Sigma, G4171), rabbit anti-hexokinase (1:1000 (IB), Cell Signaling, 2024S), rabbit anti-HIP (1:1000 (IB), Stressgen, adi-spa-766), rabbit anti-HOP (1:1000 (IB), Enzo, adi-sra-1500), mouse IgM anti-Hsc70 (13D3) (1:3000 (IB) and 1:200 (LE-IF), Novus, nb120–2788), rabbit anti-hsp40 (1:1000 (IB), Assay Designs, adi-spa-410), rabbit anti-hsp90 (1:1000 (IB), Stressgen, ADI-SPA-835-F), rat anti-LAMP-1 (1D4B) (1:3000 (IB) 1:200 (LE-IF) 1:2000 (Cell-IF), Hybridoma Bank, 1D4B, RRID AB_2134500), mouse-anti LAMP-1 (LyC16) (1:1000 (IB) and 1:100 (LE-IF), Enzo, adi-vam-en001), rabbit anti-LAMP-2A (1:5000 (IB), ThermoFisher Scientific, 512200, RRID AB_2533900) rabbit anti-LC3 (1:1000 (IB), Cell Signaling, 2775, RRID AB_915950), rabbit anti-LRRK2 (MJFF3) (1:1000 (IB), Abcam, ab133475), rabbit anti-mTOR (1:2000 (IB), Cell Signaling, 2972), mouse anti-p62 (1:1000 (IB), Abcam, ab56416), rabbit anti-Rab7 (D95F2) (1:500 (IB), Cell Signaling, 9367), mouse anti-total Tau (DA9) (1:3000 (IB) and 1:200 (LE-IF), gift from Dr. Peter Davies), rabbit anti-Tsg101 (1:1000 (IB) and 1:100 (LE-IF), Abcam, ab30871), mouse anti-tubulin (1:1000 (IB), Sigma, T7451), rabbit anti-ubiquitin (1:1000 for IB and for Cell-IF, Dako, Z0458, RRID AB_2315524), mouse anti-Vps4 (E-8) (1:500 (IB) and 1:100 (LE-IF), Santa Cruz, sc-133122), rabbit anti-Hsc70 (1μg (IP), Enzo, ADI-SPA-757), rabbit normal IgG (1μg (IP), Cell Signaling, 2729S).

Techniques: Recombinant, Injection, Western Blot, Cell Culture, Stripping Membranes, Quantitative Proteomics, Multiplex sample analysis, Plasmid Preparation, shRNA, Software, Fluorescence, Microscopy

Journal: Cell reports

Article Title: Molecular determinants of the crosstalk between endosomal microautophagy and chaperone-mediated autophagy

doi: 10.1016/j.celrep.2023.113529

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: The dilutions and sources of antibodies used for immunoblot (IB), immunoprecipitation (IP), cell immunofluorescence (cell-IF) and isolated LE/MVB immunofluorescence (LE-IF) in this study were as follows: (dilutions, commercial source, and catalog number indicated in parentheses): rabbit anti-aldolase (1:1000 (IB), Cell Signaling, 3188S), rabbit anti-Alix (1:500 (IB), Novus, NBP1–90201), mouse anti-Alix (3A9) (1:100 (LE-IF), Cell Signaling, 2171S), mouse anti-α synuclein (211) (1:2000 (IB), Santa Cruz, sc-12767), rabbit anti-ATP6V1B (1:1000 (IB), Abcam, ab200839), rabbit anti-Bag1 (1:1000 (IB), Abcam, ab32109), rabbit anti-Bag3 (1:1000 (IB), Novus, NBP2–27398SS), sheep anti-Bag6 (1:200 (IB) and 1:100 (LE-IF), R&D Systems, AF6438), rabbit anti-Bag6 (1:1000 (IB), 1:100 (LE-IF), 1μg (IP), Abcam, ab137076). rabbit anti-Cathepsin B (1:1000 (IB), Abcam, ab214428), mouse anti-cathepsin D (D-7) (1:500 (IB), Santa Cruz, sc-377299), rabbit anti-cathepsin L (H-80) (1:1000 (IB), Santa Cruz, sc-10778), rabbit anti-CHIP (C3B6) (1:1000 (IB), Cell Signaling, 2080), rabbit anti-Cyclophilin A (1:1000 (IB), Cell Signaling, 2175), mouse anti-EEA1 (1:1000 (IB), BD Biosciences, 610456, RRID AB_397830), rabbit anti-GAPDH (1:2000 (IB), Cell Signaling, 2118S), rabbit anti-GBA (1:1000 (IB), Sigma, G4171), rabbit anti-hexokinase (1:1000 (IB), Cell Signaling, 2024S), rabbit anti-HIP (1:1000 (IB), Stressgen, adi-spa-766), rabbit anti-HOP (1:1000 (IB), Enzo, adi-sra-1500), mouse IgM anti-Hsc70 (13D3) (1:3000 (IB) and 1:200 (LE-IF), Novus, nb120–2788), rabbit anti-hsp40 (1:1000 (IB), Assay Designs, adi-spa-410), rabbit anti-hsp90 (1:1000 (IB), Stressgen, ADI-SPA-835-F), rat anti-LAMP-1 (1D4B) (1:3000 (IB) 1:200 (LE-IF) 1:2000 (Cell-IF), Hybridoma Bank, 1D4B, RRID AB_2134500), mouse-anti LAMP-1 (LyC16) (1:1000 (IB) and 1:100 (LE-IF), Enzo, adi-vam-en001), rabbit anti-LAMP-2A (1:5000 (IB), ThermoFisher Scientific, 512200, RRID AB_2533900) rabbit anti-LC3 (1:1000 (IB), Cell Signaling, 2775, RRID AB_915950), rabbit anti-LRRK2 (MJFF3) (1:1000 (IB), Abcam, ab133475), rabbit anti-mTOR (1:2000 (IB), Cell Signaling, 2972), mouse anti-p62 (1:1000 (IB), Abcam, ab56416), rabbit anti-Rab7 (D95F2) (1:500 (IB), Cell Signaling, 9367), mouse anti-total Tau (DA9) (1:3000 (IB) and 1:200 (LE-IF), gift from Dr. Peter Davies), rabbit anti-Tsg101 (1:1000 (IB) and 1:100 (LE-IF), Abcam, ab30871), mouse anti-tubulin (1:1000 (IB), Sigma, T7451), rabbit anti-ubiquitin (1:1000 for IB and for Cell-IF, Dako, Z0458, RRID AB_2315524), mouse anti-Vps4 (E-8) (1:500 (IB) and 1:100 (LE-IF), Santa Cruz, sc-133122), rabbit anti-Hsc70 (1μg (IP), Enzo, ADI-SPA-757), rabbit normal IgG (1μg (IP), Cell Signaling, 2729S).

Techniques: Recombinant, Injection, Western Blot, Cell Culture, Stripping Membranes, Quantitative Proteomics, Multiplex sample analysis, Plasmid Preparation, shRNA, Software, Fluorescence, Microscopy

Journal: Journal of Biological Chemistry

Article Title: A molecular triage process mediated by RING finger protein 126 and BCL2-associated athanogene 6 regulates degradation of G0/G1 switch gene 2

doi: 10.1074/jbc.ra119.008544

Figure Lengend Snippet: Figure 6. (A) In vitro ubiquitination assay in the presence or absence of purified recombinant UBE1 (E1), UBCH5B (E2), GST-RNF126 (E3) and BAG6, together with His-tagged ubiquitin, ATP and HA-G0S2 as a substrate. The mixture was incubated for 60 min at 37oC, and the reaction was stopped by the addition of 3x sample buffer. See also Fig. S4. (B) In vitro ubiquitination assay as in (A) except for the use of GST-RNF126 WT or C231A/C234A (CA) mutant, and HA-G0S2 WT, E44A, or lysine-less mutant (6KR).

Article Snippet: The plasmid encoding human BAG6 (pRK5-FLAG-BAG6) was obtained from Addgene (#61836). siRNA library screening C2C12 cell lines stably expressing EGFP fused G0S2 or CL1 degron for siRNA library screening were generated as follows: lentiviral particles encoding EGFP-G0S2 or EGFP-CL1 (ACKNWFSSLSHFVIHL) were generated as described above, and then infected into C2C12 cells, followed by puromycin selection (5 μg/ml: SigmaAldrich).

Techniques: In Vitro, Ubiquitin Proteomics, Purification, Recombinant, Incubation, Mutagenesis