Journal: Current biology : CB

Article Title: Force-induced dephosphorylation activates the cochaperone BAG3 to coordinate protein homeostasis and membrane traffic.

doi: 10.1016/j.cub.2024.07.088

Figure Lengend Snippet: Figure 3. BAG3 interacts with RAB GTPases in a dephosphorylation- dependent manner (A) Phosphosite mutant variants of BAG3 were transiently expressed in A7r5 cells as FLAG-epitope tagged proteins followed by anti-FLAG immunopre- cipitation and mass spectrometry. (B) HSC70 was detected in complexes of wild-type BAG3, and BAG3-T285A/ S289A and BAG3-T285D/S289D mutant variants by interaction proteomics. HSPB1 was significantly more abundant (LIMMA-moderated t test p value < 0.05) in pull-downs with the dephosphorylation-mimicking BAG3- T285A/S289A. Heatmap shows protein abundance as log2-transformed mean intensity compared to mean background intensity (FLAG-BAG3- construct/empty). See Data S1A for more information. (C) Principal-component analysis based on label-free quantification (LFQ) in- tensities of 42 proteins identified as differentially abundant (ANOVA p value < 0.05 with Tukey’s post hoc test FDR < 0.05). Empty, empty vector transfected controls (black); WT, wild-type BAG3 (green); DD, BAG3-T285D/ S289D (blue); AA, BAG3-T285A/S289A (red). See Data S1B for more infor- mation. (D) Gene Ontology (GO) terms enriched among proteins identified as BAG3 interactors. GO terms were selected from significantly enriched categories (FDR-corrected p value < 0.05). For GO-term identifier numbers, see Data S1C. (E) Volcano plot visualizing the log2 transformed ratio of mean protein abundance in pull-downs with BAG3-T285A/S289A compared to BAG3- T285D/S289D (log2 (AA/DD)), plotted against log10 (LIMMA-moderated t

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit polyclonal anti-BAG3 Proteintech Cat#: 10599-1-AP; RRID: AB_2062602 Rabbit polyclonal anti-BAG3 Arndt et al.12 N/A Rabbit polyclonal anti-BAG3-pS136 This paper N/A Rabbit polyclonal anti-HSC70 Ulbricht et al.9 N/A Rabbit polyclonal anti-HSPB1 Enzo Cat#: ADI-SPA-801; RRID: AB_10615795 Rabbit polyclonal anti-HSPB8 St John s Lab Cat#: STJ24102 Rabbit polyclonal anti-LC3B Novus Biologicals Cat#: NB100-2220; RRID: AB_10003146 Rabbit polyclonal anti-RAB2A Proteintech Cat#: 15420-1-AP; RRID: AB_2176874 Rabbit polyclonal anti-RAB7A T. Watts (University of Toronto, Canada) N/A Rabbit polyclonal anti-RAB11A Abcam Cat#: Ab128913; RRID: AB_11140633 Rabbit polyclonal anti-RAB11B Proteintech Cat#: 19742-1-AP; RRID: AB_10642010 Guinea pig polyclonal anti-SQSTM1/p62 Progen Cat#: GP62-C; RRID: AB_2687531 Rabbit polyclonal anti-SYNPO2 Linnemann et al.87 N/A Mouse monoclonal anti-T7 Sigma-Aldrich Cat#: 69522 Mouse monoclonal anti-g-tubulin Sigma-Aldrich Cat#: T5326; RRID: AB_532292 Rabbit polyclonal anti-ubiquitin-pS65 Cell Signaling Cat#: 62802; RRID: AB_2799632 Bacterial and virus strains Escherichia coli BL21(DE3) Thermo Fisher Cat#: ECO114 Biological samples Human vastus lateralis muscle biopsies This paper N/A Chemicals, peptides, and recombinant proteins Cell lysis buffer Cell Signaling Technology Cat#: 9803 Protease-Inhibitor-Cocktail Thermo Fisher Cat#: 78429 Complete protease inhibitor Roche Cat#: 4693132001 PhosSTOP phosphatase inhibitor Roche Cat#: 4906837001 RIPA buffer Thermo Fisher Cat#: 89901 Protease inhibitor cocktail Sigma-Aldrich Cat#: P8340 Phosphatase inhibitor cocktail Sigma-Aldrich Cat#: P5726 M2-agarose Sigma-Aldrich Cat#: A2220 Cycloheximide Roth Cat#: 8682.1 Anti-FLAG magnetic beads Thermo Fisher Cat#: A36797 SDS, proteomics grade VWR Cat#: M112 Bafilomycin A1 Santa Cruz Biotechnology Cat#: sc-201550A Fluoromount-G Thermo Fisher Cat#: 00-4958-02 Ni-NTA-agarose Qiagen Cat#: 30210 GDP Merck Cat#: 20-177 GTP-g-S Merck Cat#: 20-176 Protein G sepharose Sigma-Aldrich Cat#: GE17-0618-01 (Continued on next page) Current Biology 34, 1–14.e1–e9, September 23, 2024 e1 Article

Techniques: De-Phosphorylation Assay, Phospho-proteomics, Mutagenesis, FLAG-tag, Mass Spectrometry, Quantitative Proteomics, Transformation Assay, Construct, Plasmid Preparation, Transfection

Journal: Current biology : CB

Article Title: Force-induced dephosphorylation activates the cochaperone BAG3 to coordinate protein homeostasis and membrane traffic.

doi: 10.1016/j.cub.2024.07.088

Figure Lengend Snippet: Figure 6. CMT2-causing RAB7A-L129F induces an overactivation of CASA in patient cells (A) Schematic presentation of the altered GTPase cycle of RAB7A-L129F as determined by McCray et al.61 RAB7A activity is controlled by a guanine nucleotide exchange factor (GEF) and a GTPase-activating protein (GAP). (B) Lymphoid cells of a control subject and CMT2 patient were treated with solvent () or BafA1 (+) for 7 h. Lysates were probed with specific antibodies against the indicated proteins. Multiple isoforms of SYNPO2 are detectable in this cell type. (C) Data obtained under (B) were quantified. Protein levels observed in solvent- treated control cells were set to 1 and were compared to levels in solvent- treated patient cells to determine changes in the steady-state levels. The dashed line indicates the control value. Data are shown as mean values ± SEM, n = 4 with two biological replicates performed in each independent experi- ment. (D) Data obtained under (B) were quantified. The ratio of protein levels with and without BafA1 treatment (ratio ± BafA1) was determined. The dashed line in- dicates a ratio of 1 with no changes upon BafA1 treatment. Data are shown as mean values ± SEM, n = 4 with two biological replicates performed in each independent experiment. (E) Schematic presentation of the stress-dependent regulation of BAG3. Statistical analysis was carried out using two-tailed unpaired t test with Welch’s correction: *p < 0.05; ns, non-significant.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit polyclonal anti-BAG3 Proteintech Cat#: 10599-1-AP; RRID: AB_2062602 Rabbit polyclonal anti-BAG3 Arndt et al.12 N/A Rabbit polyclonal anti-BAG3-pS136 This paper N/A Rabbit polyclonal anti-HSC70 Ulbricht et al.9 N/A Rabbit polyclonal anti-HSPB1 Enzo Cat#: ADI-SPA-801; RRID: AB_10615795 Rabbit polyclonal anti-HSPB8 St John s Lab Cat#: STJ24102 Rabbit polyclonal anti-LC3B Novus Biologicals Cat#: NB100-2220; RRID: AB_10003146 Rabbit polyclonal anti-RAB2A Proteintech Cat#: 15420-1-AP; RRID: AB_2176874 Rabbit polyclonal anti-RAB7A T. Watts (University of Toronto, Canada) N/A Rabbit polyclonal anti-RAB11A Abcam Cat#: Ab128913; RRID: AB_11140633 Rabbit polyclonal anti-RAB11B Proteintech Cat#: 19742-1-AP; RRID: AB_10642010 Guinea pig polyclonal anti-SQSTM1/p62 Progen Cat#: GP62-C; RRID: AB_2687531 Rabbit polyclonal anti-SYNPO2 Linnemann et al.87 N/A Mouse monoclonal anti-T7 Sigma-Aldrich Cat#: 69522 Mouse monoclonal anti-g-tubulin Sigma-Aldrich Cat#: T5326; RRID: AB_532292 Rabbit polyclonal anti-ubiquitin-pS65 Cell Signaling Cat#: 62802; RRID: AB_2799632 Bacterial and virus strains Escherichia coli BL21(DE3) Thermo Fisher Cat#: ECO114 Biological samples Human vastus lateralis muscle biopsies This paper N/A Chemicals, peptides, and recombinant proteins Cell lysis buffer Cell Signaling Technology Cat#: 9803 Protease-Inhibitor-Cocktail Thermo Fisher Cat#: 78429 Complete protease inhibitor Roche Cat#: 4693132001 PhosSTOP phosphatase inhibitor Roche Cat#: 4906837001 RIPA buffer Thermo Fisher Cat#: 89901 Protease inhibitor cocktail Sigma-Aldrich Cat#: P8340 Phosphatase inhibitor cocktail Sigma-Aldrich Cat#: P5726 M2-agarose Sigma-Aldrich Cat#: A2220 Cycloheximide Roth Cat#: 8682.1 Anti-FLAG magnetic beads Thermo Fisher Cat#: A36797 SDS, proteomics grade VWR Cat#: M112 Bafilomycin A1 Santa Cruz Biotechnology Cat#: sc-201550A Fluoromount-G Thermo Fisher Cat#: 00-4958-02 Ni-NTA-agarose Qiagen Cat#: 30210 GDP Merck Cat#: 20-177 GTP-g-S Merck Cat#: 20-176 Protein G sepharose Sigma-Aldrich Cat#: GE17-0618-01 (Continued on next page) Current Biology 34, 1–14.e1–e9, September 23, 2024 e1 Article

Techniques: Activity Assay, Control, Solvent, Two Tailed Test

Journal: Intervirology

Article Title: The Regulation of Prototype Foamy Virus 5′Long Terminal Repeats and Internal Promoter by Endogenous Transcription Factors

doi: 10.1159/000517539

Figure Lengend Snippet: Plasmids used in the assays

Article Snippet: Then the proteins were transferred to PVDF membrane and incubated with anti-BAG3 (Bioss, China) and anti-fos (Bioss) polyclonal antibodies, respectively.

Techniques: Plasmid Preparation, Expressing

Journal: Intervirology

Article Title: The Regulation of Prototype Foamy Virus 5′Long Terminal Repeats and Internal Promoter by Endogenous Transcription Factors

doi: 10.1159/000517539

Figure Lengend Snippet: Promoter activities of PFV 5′LTR and IP regulated by AP-1 (a) and BAG3 (b). The plasmids pcDNA-jun and pcDNA-fos were used to express the 2 subunits of the heterodimer AP-1. The plasmid pcDNA-BAG3 was used to express the BAG3 protein. At the bottom of each column, the amount (nanogram) of the plasmids co-transfected in each group was labeled. PFV, prototype foamy virus; LTR, long terminal repeat; IP, internal promoter; TRE, Tas responsive elements; AP-1, activator promoter-1; PMA, phorbol-12-myristate-13-acetate; BAG3, BCL2-associated athanogene 3. Asterisks indicate significant difference.

Article Snippet: Then the proteins were transferred to PVDF membrane and incubated with anti-BAG3 (Bioss, China) and anti-fos (Bioss) polyclonal antibodies, respectively.

Techniques: Plasmid Preparation, Transfection, Labeling