anti bag3  (Cell Signaling Technology Inc)

Journal: Life Science Alliance

Article Title: Identification of phosphatases that dephosphorylate the co-chaperone BAG3

doi: 10.26508/lsa.202402734

Figure Lengend Snippet: (A) Linear illustration of human BAG3 protein sequence with reported p-sites annotated. (B) Co-IP of FLAG-BAG3 from HEK293 cells revealed significantly enriched proteins, assessed using the rankprod method (n = 4), with a minimum fivefold increase and a P -value ≤ 0.05. Enriched proteins in the sample are marked in respective colors for subcategories. Selected hits are annotated with their gene name. A false discovery rate of 5% is indicated by a dashed line. (C) Enriched phosphatases and respective phosphatase regulators from the BAG3 co-IP sample were assigned to their respective (super-)families of human phosphatases. The phosphoprotein phosphatase family is colored; other Ser/Thr-specific phosphatases are in shades of gray. Gene names are listed next to the corresponding assigned phosphatase identifications. PPMs, metal-dependent protein phosphatases; PSTPs, protein serine/threonine-specific phosphatases. (A, D) Gene ontology enrichment analysis of significantly enriched genes from the BAG3 co-IP sample (n = 277) in (A). The 10 most abundant biological process GO terms are displayed as bars, ranked based on protein counts. Biological processes related to BAG3’s role in protein homeostasis are presented in blue. (E) Gene ontology enrichment analysis of the co-IP sample compared with overall human gene expression, displayed as a heatmap. Fold enrichment was calculated for the BAG3 enriched, not enriched, and total co-IP sets, with overall GO categories annotated at the respective cluster. Statistical significance was determined using Fisher’s test ( P -value < 0.05) with the Bonferroni correction implemented in the PANTHER database. Non-significant changes are colored gray within the heatmap.

Article Snippet: The membranes were blocked with Tris-buffered saline with 0.1% Tween (TBS-T) containing 5% (wt/vol) non-fat milk at RT for 60 min. After three washes (5 min), the membranes were incubated overnight at 4°C with the following antibodies in TBS-T with 5% BSA: α-GAPDH (1:1,000, #2118), α-BAG3 (1:1,000, 23842S), and α-PP5 (1:1,000, 2289S), α-CDC37 (1:1,000, 3618S), α-CDC37-pS13 (1:1,000, 13,248), α-phosphoThr (1:500, 42H4, 9386), SQSTM1/p62 (1:1,000, 5114T), and α-14-3-3γ (1:500, 5522S) obtained from Cell Signaling Technologies.

Techniques: Sequencing, Co-Immunoprecipitation Assay, Gene Expression

Journal: Life Science Alliance

Article Title: Identification of phosphatases that dephosphorylate the co-chaperone BAG3

doi: 10.26508/lsa.202402734

Figure Lengend Snippet: (A) Illustration of the used FLAG-control construct to determine unspecific binding and the N -terminally triple-FLAG–tagged human BAG3 used as the bait for the FLAG co-IP. (B) A schematic overview displays annotated BAG3 domains for orientation. BAG3 sequences available from public databases (377 species, all vertebrates) were aligned by geneious using the MUSCLE algorithm. A heat map representation of the alignment identity score was generated to visualize the sequence conservation of BAG3. Regions in the alignment corresponding to gaps in the reference (BAG3 of homo sapiens ) were excluded. (C) The multiple sequence alignment for the p-site stretches of interest in this study are underlined and represented in more detail using sequence logos (see (C)). (B, C) Phylogenetic analysis of the multiple sequence alignment displayed in (B). A consensus tree was built with Jukes-Contor model and Neighbor-Joining method after 200 bootstrap replications. The consensus tree for the 377 BAG3 orthologs reveals three primary clusters, corresponding to Teleostei, Sarcopterygii, and mammals. Sequence logos display the context of BAG3-pS136 and the p-site cluster (pS284-pS291) phosphorylation sites for these three principal orthologous groups. Phosphorylation sites are not conserved across all vertebrate classes, but they are conserved in mammals. In the tree, the BAG3 positions of mouse, rat, and human are indicated by black triangles, arranged from left to right.

Article Snippet: The membranes were blocked with Tris-buffered saline with 0.1% Tween (TBS-T) containing 5% (wt/vol) non-fat milk at RT for 60 min. After three washes (5 min), the membranes were incubated overnight at 4°C with the following antibodies in TBS-T with 5% BSA: α-GAPDH (1:1,000, #2118), α-BAG3 (1:1,000, 23842S), and α-PP5 (1:1,000, 2289S), α-CDC37 (1:1,000, 3618S), α-CDC37-pS13 (1:1,000, 13,248), α-phosphoThr (1:500, 42H4, 9386), SQSTM1/p62 (1:1,000, 5114T), and α-14-3-3γ (1:500, 5522S) obtained from Cell Signaling Technologies.

Techniques: Control, Construct, Binding Assay, Co-Immunoprecipitation Assay, Generated, Sequencing, Phospho-proteomics

Journal: Life Science Alliance

Article Title: Identification of phosphatases that dephosphorylate the co-chaperone BAG3

doi: 10.26508/lsa.202402734

Figure Lengend Snippet: (A) HEK293 cells and A7r5 smooth muscle cells were lysed, and protein levels of BAG3 were assessed in relation to the housekeeping protein GAPDH using immunoblots. Quantitative analysis of BAG3 expression validated elevated expression in A7r5 cells. Statistical significance was evaluated through t tests, incorporating data from eight independent replicates. (B) Alignment of human, mouse, and rat BAG3 orthologs to visualize the conservation of the p-sites of interest. Functional p-site studies were carried out in these three organisms in preliminary studies and in rat and human in this work. (C) Summary of the phosphopeptides used in enzyme kinetic studies for the assessment of PP1c and PP5c-mediated dephosphorylation of BAG3 mimetics. (D) Monitoring of CDC37-pSer13 dephosphorylation in A7r5 lysate upon incubation with PP5, for the indicated incubation time to validate enzymatic activity. Endogenous phosphatases were inhibited by the addition of 20 nM CalA. Quantification depicts the results of five independent experiments with adjusted P -values obtained from one-way ANOVA with Sidak correction (*** P = 0.0004), **** P < 0.0001). Mean is shown and error bars represent the SD. Source data are available for this figure.

Article Snippet: The membranes were blocked with Tris-buffered saline with 0.1% Tween (TBS-T) containing 5% (wt/vol) non-fat milk at RT for 60 min. After three washes (5 min), the membranes were incubated overnight at 4°C with the following antibodies in TBS-T with 5% BSA: α-GAPDH (1:1,000, #2118), α-BAG3 (1:1,000, 23842S), and α-PP5 (1:1,000, 2289S), α-CDC37 (1:1,000, 3618S), α-CDC37-pS13 (1:1,000, 13,248), α-phosphoThr (1:500, 42H4, 9386), SQSTM1/p62 (1:1,000, 5114T), and α-14-3-3γ (1:500, 5522S) obtained from Cell Signaling Technologies.

Techniques: Western Blot, Expressing, Functional Assay, De-Phosphorylation Assay, Incubation, Activity Assay

Journal: Life Science Alliance

Article Title: Identification of phosphatases that dephosphorylate the co-chaperone BAG3

doi: 10.26508/lsa.202402734

Figure Lengend Snippet: (A) A7r5 smooth muscle cells were treated with increasing concentrations of phosphoprotein phosphatase inhibitors CalA or OA as indicated, or DMSO as control. After 20 min of incubation, cells were lysed, and modulator effects on BAG3-pS136 were determined with immunoblots. The quantification is presented as a bar plot, with the mean depicted with error bars that represent the SEM based on five independent experiments. Statistical significance between inhibitors was determined with t test and P -values are displayed as stars (* P = 0.020, *** P < 0.001). (B, C) Michaelis–Menten kinetic parameters of PP1c were determined against the indicated mono- or bisphosphorylated peptides. Error bars represent the SEM of three independent replicates with technical duplicates. k cat / K m was calculated by comparison with a phosphate standard curve. (D) Overexpressed FLAG-BAG3 from transiently transfected HEK293 cells was single-step affinity immobilized with anti-FLAG beads and treated with PP1c for 30 min. Immunoblot quantification depicts the mean of four independent experiments, the error bar represents the SD, with P -values obtained from a t tests (two-tailed, paired, **** P < 0.001). (E) Monitoring of BAG3-pS136 dephosphorylation in A7r5 lysate upon incubation with PP1c, PP5, or without phosphatase as a control treatment for the indicated incubation time. Endogenous phosphatases were inhibited by the addition of 20 nM CalA. Quantification depicts the results of four independent experiments with P -values obtained from two-way ANOVA with Sidak correction (** P = 0.006 [PP1c], ** P = 0.005 [PP5], **** P < 0.001). Mean is shown and error bars represent the SEM. Source data are available for this figure.

Article Snippet: The membranes were blocked with Tris-buffered saline with 0.1% Tween (TBS-T) containing 5% (wt/vol) non-fat milk at RT for 60 min. After three washes (5 min), the membranes were incubated overnight at 4°C with the following antibodies in TBS-T with 5% BSA: α-GAPDH (1:1,000, #2118), α-BAG3 (1:1,000, 23842S), and α-PP5 (1:1,000, 2289S), α-CDC37 (1:1,000, 3618S), α-CDC37-pS13 (1:1,000, 13,248), α-phosphoThr (1:500, 42H4, 9386), SQSTM1/p62 (1:1,000, 5114T), and α-14-3-3γ (1:500, 5522S) obtained from Cell Signaling Technologies.

Techniques: Control, Incubation, Western Blot, Comparison, Transfection, Two Tailed Test, De-Phosphorylation Assay

Journal: Life Science Alliance

Article Title: Identification of phosphatases that dephosphorylate the co-chaperone BAG3

doi: 10.26508/lsa.202402734

Figure Lengend Snippet: (A) Illustrative overview of the small molecule and peptide modulator screen to tune PP1 activity in cells. HEK293 cells transiently expressing FLAG-BAG3 were treated as illustrated, lysed, and captured by a single-step affinity enrichment using anti-FLAG beads. Samples were analyzed by immunoblots. Quantification depicts results of three independent experiments with P -values obtained from t tests (two-tailed). Mean is shown with replicates as scatter plot and error bars represent the SD (* P = 0.015, *** P < 0.001). (B) A7r5 smooth muscle cells were incubated with increasing concentrations of Tautomycetin as indicated. Immunoblots of lysate were used to determine the sensitivity of the titrated inhibitor towards BAG3-pS136. Mean is shown as a barplot with replicates as scatter plots, and error bars represent the SD of four independent experiments. Statistical significance between concentrations is determined with t test with Welch’s correction (* P < 0.05). (C) Analysis of phosphorylation-dependent binding of 14-3-3γ to FLAG-BAG3 in A7r5 muscle cells after 20-min modulation of endogenous PP1 before lysis and FLAG-IP enrichment. Results are presented as a barplot, P -values obtained from a t test (*** P = 0.0002, **** P < 0.0001). (D) Immunoblots depicting siRNA-mediated knockdown of all PP1 isoforms combined (PP1α/β/γ) or individual PP1 isoforms (PP1α, PP1β, PP1γ) separately. The displayed blots represent the analyzed protein levels of 24 or 48 h after transfection. (E) Quantification of immunoblotted lysates show level changes of PP1α/β/γ and BAG3 (n = 3 or 4). Quantification depicts results of three or four independent experiments with P -values obtained from t tests (two-tailed, paired). Error bars represent the SD. Source data are available for this figure.

Article Snippet: The membranes were blocked with Tris-buffered saline with 0.1% Tween (TBS-T) containing 5% (wt/vol) non-fat milk at RT for 60 min. After three washes (5 min), the membranes were incubated overnight at 4°C with the following antibodies in TBS-T with 5% BSA: α-GAPDH (1:1,000, #2118), α-BAG3 (1:1,000, 23842S), and α-PP5 (1:1,000, 2289S), α-CDC37 (1:1,000, 3618S), α-CDC37-pS13 (1:1,000, 13,248), α-phosphoThr (1:500, 42H4, 9386), SQSTM1/p62 (1:1,000, 5114T), and α-14-3-3γ (1:500, 5522S) obtained from Cell Signaling Technologies.

Techniques: Activity Assay, Expressing, Western Blot, Two Tailed Test, Incubation, Phospho-proteomics, Binding Assay, Lysis, Knockdown, Transfection

Journal: Life Science Alliance

Article Title: Identification of phosphatases that dephosphorylate the co-chaperone BAG3

doi: 10.26508/lsa.202402734

Figure Lengend Snippet: (A) Schematic illustration of the protein level assessment after treatment of HEK293 cells with pre-designed siRNA. HEK293 cells were transfected with siRNA of PP1 isoforms (PPP1CA/B/C) for 24 and 48 h. (B) Quantification of immunoblotted lysates show phosphorylation level changes of BAG3-pS136 relative to the overall BAG3 levels (n = 3 or 4). Quantification depicts results of three or four independent experiments with P -values obtained from t tests (two-tailed, paired). Error bars represent the SD. Source data are available for this figure.

Article Snippet: The membranes were blocked with Tris-buffered saline with 0.1% Tween (TBS-T) containing 5% (wt/vol) non-fat milk at RT for 60 min. After three washes (5 min), the membranes were incubated overnight at 4°C with the following antibodies in TBS-T with 5% BSA: α-GAPDH (1:1,000, #2118), α-BAG3 (1:1,000, 23842S), and α-PP5 (1:1,000, 2289S), α-CDC37 (1:1,000, 3618S), α-CDC37-pS13 (1:1,000, 13,248), α-phosphoThr (1:500, 42H4, 9386), SQSTM1/p62 (1:1,000, 5114T), and α-14-3-3γ (1:500, 5522S) obtained from Cell Signaling Technologies.

Techniques: Transfection, Phospho-proteomics, Two Tailed Test

Journal: Life Science Alliance

Article Title: Identification of phosphatases that dephosphorylate the co-chaperone BAG3

doi: 10.26508/lsa.202402734

Figure Lengend Snippet: (A) Phosphorylation levels of BAG3-pS136 were compared in HEK293 cells overexpressing FLAG-BAG3 or both FLAG-BAG3 and FLAG-PP5 for 24 h. Immunoblot quantification demonstrates that PP5 overexpression does not affect BAG3-pS136 phosphorylation. Statistical significance was determined through t test (n = 4). (B) Michaelis–Menten kinetic parameters of PP5c were determined against the indicated mono- or bisphosphorylated peptides. Error bars represent the SEM of three independent repeats with technical duplicates. k cat / K m was calculated by comparison with a phosphate standard curve. (C) HEK293 cells overexpressed FLAG-BAG3 and were treated with 1 μM recombinant PP5 after lysis for 1 h (in vitro), or overexpressed FLAG-BAG3 and FLAG-PP5 simultaneously for 24 h (in-cell), or were not treated with phosphatase (Ctrl). FLAG-BAG3 was subjected to affinity enrichment and subsequently evaluated using LC-MS/MS. The theoretical phosphopeptide generated through trypsinization is shown at the top. Targeted phosphoproteomics quantification reveals the complete dephosphorylation of the p-site cluster for both PP5 treatments. Statistical significance was determined through two-way ANOVA (n = 4), error bars represent the SD (**** P < 0.0001, *** P = 0.0002, * P = 0.0021). Indexed retention time (iRT). (D) FLAG-BAG3 mutants mimicking the phosphorylated (4D) and dephosphorylated (4A) state of the BAG3 p-site cluster were overexpressed in HEK293 cells followed by co-IP and MS analysis to assess PP5 binding to each variant compared with WT. Bars represent the mean (n = 3), with significance and error bars calculated using t test (* P = 0.03). (C, E) Samples were subjected to the same treatment as described for (C) to induce dephosphorylation of p-site cluster. This was followed by the analysis of protein quantities bound to BAG3 via immunoblotting, normalized to the amounts bound to the control sample. Statistical significance of changes in protein levels was assessed using data from seven or nine independent replicates, with means presented as bar plots. P -values were calculated using a one-way ANOVA with a 95% confidence interval and analyzed using PRISM/GraphPad version 6 (HspB8: * P = 0.0188; ** P = 0.0002; 14-3-3: P [in vitro] = 0.1668, P [in-cell] = 0.6241). (F) A7r5 cells were transfected with PPP5C siRNA for the specified incubation time and protein levels were determined using immunoblots. Statistical significance in the changes of protein levels was determined based on data from five independent replicates, and the means are presented as bar plots. The P -value was calculated using a two-tailed test with a 95% confidence interval and determined using PRISM/GraphPad version 6 (PP5: *** P = 0.001, ** P = 0.0045, *** P = 0.0006; BAG3: ** P = 0.0097, * P = 0.046, * P = 0.0328). (G) Smooth muscle cells were subjected to treatment with control siRNA or PP5-targeting siRNA (siPP5) followed by treatment with BafA1 to inhibit CASA mediated BAG3 degradation. Statistical significance of changes in protein levels was assessed using data from seven independent replicates, represented as boxplots. P -values were calculated using a two-way ANOVA and analyzed using PRISM/GraphPad version 6. Source data are available for this figure.

Article Snippet: The membranes were blocked with Tris-buffered saline with 0.1% Tween (TBS-T) containing 5% (wt/vol) non-fat milk at RT for 60 min. After three washes (5 min), the membranes were incubated overnight at 4°C with the following antibodies in TBS-T with 5% BSA: α-GAPDH (1:1,000, #2118), α-BAG3 (1:1,000, 23842S), and α-PP5 (1:1,000, 2289S), α-CDC37 (1:1,000, 3618S), α-CDC37-pS13 (1:1,000, 13,248), α-phosphoThr (1:500, 42H4, 9386), SQSTM1/p62 (1:1,000, 5114T), and α-14-3-3γ (1:500, 5522S) obtained from Cell Signaling Technologies.

Techniques: Phospho-proteomics, Western Blot, Over Expression, Comparison, Recombinant, Lysis, In Vitro, Liquid Chromatography with Mass Spectroscopy, Generated, De-Phosphorylation Assay, Co-Immunoprecipitation Assay, Binding Assay, Variant Assay, Control, Transfection, Incubation, Two Tailed Test

Journal: Life Science Alliance

Article Title: Identification of phosphatases that dephosphorylate the co-chaperone BAG3

doi: 10.26508/lsa.202402734

Figure Lengend Snippet: (A) Schematic representation of the experimental procedure used for the dephosphorylation analysis of the p-site cluster of FLAG-BAG3 derived from HEK293 cells. Cells were either overexpressing FLAG-PP5 or subjected to in vitro PP5 incubation. This workflow served to decipher the dephosphorylation of the BAG3 p-site cluster (LC-MS/MS; ). (B) HEK293 cells overexpressing FLAG-BAG3 were subjected to various modulations of PP5 activity including PP5 co-overexpression for 24 h, activation using arachidonic acid (2 h, 250 μM), or in vitro incubation with recombinant PP5 (1 h, 1 μM, 30°C) in lysate or on-beads (30 min, 1 μM PP5, 30°C). LC-MS/MS was used to quantitatively assess the dephosphorylation status of individual p-sites within the p-site cluster. (C) Related to . Immunoblots of lysates from BAG3-overexpressing HEK293 cells that were treated with PP5 to ensure equal amounts of proteins were present. (D) Related to . Immunoblots of FLAG-BAG3 co-IP samples from HEK293 cells subjected to dephosphorylation treatments with PP5. (E) A7r5 cells were transfected with PPP5C siRNA for the specified incubation time and protein levels were determined using immunoblots. Statistical significance in the changes of protein levels was determined based on data from five independent replicates, and the means are presented as bar plots. The nonparametric Spearman correlation between BAG3 and PP5 upon PPP5C siRNA treatment verifies the negative correlation between the protein levels. (F) Smooth muscle cells were subjected to treatment with control siRNA or PP5-targeting siRNA (siPP5) followed by treatment with BafA1 to inhibit CASA-mediated degradation. Statistical significance of changes in protein levels of SQSTM1 was assessed using data from seven independent replicates, represented as boxplots. P -values were calculated using a two-way ANOVA and analyzed using PRISM/GraphPad version 6. Source data are available for this figure.

Article Snippet: The membranes were blocked with Tris-buffered saline with 0.1% Tween (TBS-T) containing 5% (wt/vol) non-fat milk at RT for 60 min. After three washes (5 min), the membranes were incubated overnight at 4°C with the following antibodies in TBS-T with 5% BSA: α-GAPDH (1:1,000, #2118), α-BAG3 (1:1,000, 23842S), and α-PP5 (1:1,000, 2289S), α-CDC37 (1:1,000, 3618S), α-CDC37-pS13 (1:1,000, 13,248), α-phosphoThr (1:500, 42H4, 9386), SQSTM1/p62 (1:1,000, 5114T), and α-14-3-3γ (1:500, 5522S) obtained from Cell Signaling Technologies.

Techniques: De-Phosphorylation Assay, Derivative Assay, In Vitro, Incubation, Liquid Chromatography with Mass Spectroscopy, Activity Assay, Over Expression, Activation Assay, Recombinant, Western Blot, Co-Immunoprecipitation Assay, Transfection, Control

Journal: Life Science Alliance

Article Title: Identification of phosphatases that dephosphorylate the co-chaperone BAG3

doi: 10.26508/lsa.202402734

Figure Lengend Snippet: Dephosphorylation of pS136 on BAG3 by PP1 leads to loss of 14-3-3 protein binding. Dephosphorylation of the p-site cluster pS284-pS291 by PP5 enables HspB8 binding, and PP5 depletion increases BAG3 levels in a CASA-dependent manner.

Article Snippet: The membranes were blocked with Tris-buffered saline with 0.1% Tween (TBS-T) containing 5% (wt/vol) non-fat milk at RT for 60 min. After three washes (5 min), the membranes were incubated overnight at 4°C with the following antibodies in TBS-T with 5% BSA: α-GAPDH (1:1,000, #2118), α-BAG3 (1:1,000, 23842S), and α-PP5 (1:1,000, 2289S), α-CDC37 (1:1,000, 3618S), α-CDC37-pS13 (1:1,000, 13,248), α-phosphoThr (1:500, 42H4, 9386), SQSTM1/p62 (1:1,000, 5114T), and α-14-3-3γ (1:500, 5522S) obtained from Cell Signaling Technologies.

Techniques: De-Phosphorylation Assay, Protein Binding, Binding Assay

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