mouse anti human cdc37 antibody  (Proteintech)

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) 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