isoform-specific pp1 antibodies α-pp1α/β/γ  (Santa Cruz Biotechnology)

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: Isoform-specific PP1 antibodies α-PP1α/β/γ (1:1,000, sc-271762/sc-365678/sc-515943) were from Santa Cruz.

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) 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: Isoform-specific PP1 antibodies α-PP1α/β/γ (1:1,000, sc-271762/sc-365678/sc-515943) were from Santa Cruz.

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: Isoform-specific PP1 antibodies α-PP1α/β/γ (1:1,000, sc-271762/sc-365678/sc-515943) were from Santa Cruz.

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: Isoform-specific PP1 antibodies α-PP1α/β/γ (1:1,000, sc-271762/sc-365678/sc-515943) were from Santa Cruz.

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: 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: Isoform-specific PP1 antibodies α-PP1α/β/γ (1:1,000, sc-271762/sc-365678/sc-515943) were from Santa Cruz.

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

Journal: BMC Cancer

Article Title: OSU-03012 sensitizes breast cancers to lapatinib-induced cell killing: a role for Nck1 but not Nck2

doi: 10.1186/1471-2407-13-256

Figure Lengend Snippet: Phosphorylation of eIF2-α indicates ER stress signaling in MDA-MB-231 and BT474 cells after treatment with OSU-03012 and lapatinib. MDA-MB-231 cells and BT474 cells (1 x 10 6 ) were subjected to vehicle (DMSO, Ctr), OSU-03012 (2 μM), lapatinib (2 μM) or the combination as indicated for 3 h. Cells were then lysed and protein extracts (10-20 μg) were subjected to SDS PAGE followed by western immunoblotting for the indicated proteins.

Article Snippet: Concurrently, Protein A/G agarose beads (Pierce) were incubated with antibodies raised against either total eIF2-α or total PP1 (Cell Signaling).

Techniques: SDS Page, Western Blot

Journal: BMC Cancer

Article Title: OSU-03012 sensitizes breast cancers to lapatinib-induced cell killing: a role for Nck1 but not Nck2

doi: 10.1186/1471-2407-13-256

Figure Lengend Snippet: The role of eIF2-α phosphorylation in cell death induced by OSU-03012 and lapatinib. A-B : MDA-MB-231 ( A ) or BT474 ( B ) cells (5 x 10 5 ) were transfected with the indicated siRNA molecules and incubated for 48 h. Cells were then treated with either vehicle (Veh) or the combination of OSU-03012 (2 μM) and lapatinib (2 μM) (combo) as indicated and either subjected immunoassays (bottom panels) or plated for clonogenic capacity (top graphs). Numbers indicated are percent control (e.g. Veh). C-D : MDA-MB-231 cells (5 x 10 5 ) were transfected with a control vector (Vector), wild-type (WT) or the Ser 51 Ala mutant (S51A) eIF2-α plasmids. After a further 24 h cells were plated onto 6-well plates to assay for clonogenic capacity ( D ) or subjected to immunoblotting as described in Materials and Methods ( C ). Cells were treated with either vehicle (Veh, DMSO) or OSU-03012 (2 μM) and lapatinib (2 μM) in combination (combo) for 24 h ( D ) or 3 h ( C ). Colonies were allowed to develop for the next 10-14 days. * indicates a p < 0.05 with respect to vehicle-treated cells. E : MDA-MB-231 cells (1 x 10 6 ) were plated and treated with the indicated drugs (Vehicle (Ctr, DMSO), OSU-03012 (OSU, 2 μM), lapatinib (Lap, 2 μM) or the combination (Combo)). Three hours later, cells were harvested and subjected to immunoblotting. Samples were probed with the indicated antibodies (see Materials and Methods).

Article Snippet: Concurrently, Protein A/G agarose beads (Pierce) were incubated with antibodies raised against either total eIF2-α or total PP1 (Cell Signaling).

Techniques: Transfection, Incubation, Plasmid Preparation, Mutagenesis, Western Blot

Journal: BMC Cancer

Article Title: OSU-03012 sensitizes breast cancers to lapatinib-induced cell killing: a role for Nck1 but not Nck2

doi: 10.1186/1471-2407-13-256

Figure Lengend Snippet: Upstream signaling responsible for eIF2-α phosphorylation: A role for Nck1. A-B : MDA-MB-231 cells and BT474 cells were treated with vehicle (Ctr, DMSO), OSU-03012 (OSU, 2 μM), lapatinib (Lap, 2 μM) or the combination (Combo) for 3 h, then harvested for immunoblotting assays as described in Materials and Methods. Membranes were probed with the indicated antibodies. C : MDA-MB-231 cells (5 x 10 5 ) were transfected with plasmids to express either GFP-Nck1 or GFP-Nck2 as described. After an additional 24 h, cells were treated with the combination of OSU-03012 and lapatinib as indicated for 24 h (upper graphs) or 3 h (lower panels), and then either plated onto 6-well dishes and allowed to form colonies (graphs represent percent control) or harvested for immunoblotting assays and probed with the indicated antibodies. * indicates a p<0.05.

Article Snippet: Concurrently, Protein A/G agarose beads (Pierce) were incubated with antibodies raised against either total eIF2-α or total PP1 (Cell Signaling).

Techniques: Western Blot, Transfection

Journal: BMC Cancer

Article Title: OSU-03012 sensitizes breast cancers to lapatinib-induced cell killing: a role for Nck1 but not Nck2

doi: 10.1186/1471-2407-13-256

Figure Lengend Snippet: Nck1, but not Nck2 expression ablates the increase in cell death induced by OSU-03012 and lapatinib. A-D : MDA-MB-231 cells ( A, B ), or BT474 cells ( C, D ) (5 x 10 5 ) were co-transfected with either wild-type eIF2-α and the two Nck isoforms ( A, C ), or the S 51 A phospho-mutant and the two Nck isoforms ( B, D ). Cells were allowed to incubate for 24 h to induce ectopic expression, and then treated with either vehicle (indicated with a -), or the combination of OSU-03012 and lapatinib (2 μM each, indicated with a +). Cells were either harvested after a 3 h treatment for western blotting as described in Materials and Methods (bottom panels), or plated singly onto 6-well plates to assay for clonogenic capacity (upper graphs). Cells were treated with OSU-03012 (2 μM) and lapatinib (2 μM) for 24 h, and then allowed to form colonies for 10-14 days. Total colony counts were graphed. * denotes a p-value of <0.05.

Article Snippet: Concurrently, Protein A/G agarose beads (Pierce) were incubated with antibodies raised against either total eIF2-α or total PP1 (Cell Signaling).

Techniques: Expressing, Transfection, Mutagenesis, Western Blot

Journal: BMC Cancer

Article Title: OSU-03012 sensitizes breast cancers to lapatinib-induced cell killing: a role for Nck1 but not Nck2

doi: 10.1186/1471-2407-13-256

Figure Lengend Snippet: Nck1 is upstream of eIF2-α phosphorylation in the cell death induced by the combination of OSU-03012 and lapatinib. A : BT474 cells (5 x 10 5 ) were co-transfected with the Ser 51 Ala eIF2-α mutant and GFP-Nck1. Cells were allowed to incubate for 24 h to induce ectopic expression then treated with either vehicle (indicated with a -), or the combination of OSU-03012 and lapatinib (2 μM each, indicated with a +). Cells were either harvested for western blotting as described previously (lower panel), or plated into 6-well plates to assay for clonogenic capacity as described previously (upper graph). Graphs represent total colony number. * denotes a p-value of <0.05. B : BT474 (upper panel) and MDA-MB-231 (lower panel) cells were treated with either vehicle (Veh) or OSU-03012 and lapatinib in combination (combo). PP1 was immunoprecipitated and the resulting membranes were immunoblotted with eIF2α and PP1. C : Our data indicate that Nck1 and PP1, which are originally in a complex with eIF2 are dissociated after treatment with the combination of OSU-03012 and lapatinib. This event frees eIF2-α to become phosphorylated by one of many upstream kinases such as PERK, leading to ER stress and eventual cell death.

Article Snippet: Concurrently, Protein A/G agarose beads (Pierce) were incubated with antibodies raised against either total eIF2-α or total PP1 (Cell Signaling).

Techniques: Transfection, Mutagenesis, Expressing, Western Blot, Immunoprecipitation

Journal: Nature Communications

Article Title: Dissecting the sequence determinants for dephosphorylation by the catalytic subunits of phosphatases PP1 and PP2A

doi: 10.1038/s41467-020-17334-x

Figure Lengend Snippet: a Scheme of the experimental PLDMS workflow. b Phosphopeptide libraries used for substrate preference evaluation. c Amino acid distribution in the permutated positions ( x1 and x2 ) of the Nterm_x1_1 (blue) and the Cterm library (red) after reference measurement (samples not treated with PP1 or PP2A, i.e. untreated) and filtering for expected sequences (see Supplementary Table and the methods section). Perfectly random incorporation of all amino acids would result in 7% per amino acid. Source data are provided as a Source Data file. d Mascot Score (statistical value for how well detected data matches database sequences) distributions of the reference measurements for Nterm_x1_1 (left) and Cterm library (right). Empirically wrong peptides are peptides with sequences not matching expected sequences from the synthetic route. For the reference measurement, empirical filtering by Mascot Score cut-offs of 39 and 32 at a false discovery rate (FDR) of 5% for the Nterm_x1_1 and the Cterm library, respectively, allowed separation of correct peptides from wrong ones.

Article Snippet: After three washes (5 min), membranes were then incubated with the following antibodies in TBS-T with 5% BSA overnight at 4 °C: α-GFP (1:2000, Abcam, ab6556), α-GAB2 (1:1000, CST, #3239), α-GAB2-pS210 (1:250, Symansis, customized purification), α-FLAG (1:1000, M2, Sigma-Aldrich, F3165), α-pan14-3-3 (1:500, SCBT, sc-629), α-PP1 (1:1000, SCBT, sc-7482), α-PP2A (1:1000, SCBT, sc-130237) or α-6His (1:2000, Abcam, ab9108).

Techniques: Phospho-proteomics

Journal: Nature Communications

Article Title: Dissecting the sequence determinants for dephosphorylation by the catalytic subunits of phosphatases PP1 and PP2A

doi: 10.1038/s41467-020-17334-x

Figure Lengend Snippet: a Workflow of phosphoproteomic experiments. b Identified p-sites were filtered for class I sites (localization probability of phosphorylation >0.75) and compared to known phosphosites from PhosphositePlus (v03/07/18) (Supplementary Data ). c Differentially downregulated p-sites between PP1 and PP2A as determined by Student’s t test (two-sided, FDR 0.01) (see Supplementary Fig. ). d PP1c and PP2Ac prefer pThr over pSer. The percentage of Thr found in PP1c/PP2Ac dephosphorylated p-sites with a log2 fold-change < −1 is significantly larger than the percentage of pThr in p-sites with a log2 fold-change > −1 according to a two-sided Fisher’s exact test (***: p -value <0.001; 1.175e–06 (PP1), 2.426e–06 (PP2A)). n.s.: not significant. Source data of Fig. 3b, d are provided as a Source Data file.

Article Snippet: After three washes (5 min), membranes were then incubated with the following antibodies in TBS-T with 5% BSA overnight at 4 °C: α-GFP (1:2000, Abcam, ab6556), α-GAB2 (1:1000, CST, #3239), α-GAB2-pS210 (1:250, Symansis, customized purification), α-FLAG (1:1000, M2, Sigma-Aldrich, F3165), α-pan14-3-3 (1:500, SCBT, sc-629), α-PP1 (1:1000, SCBT, sc-7482), α-PP2A (1:1000, SCBT, sc-130237) or α-6His (1:2000, Abcam, ab9108).

Techniques: Phospho-proteomics

Journal: Nature Communications

Article Title: Dissecting the sequence determinants for dephosphorylation by the catalytic subunits of phosphatases PP1 and PP2A

doi: 10.1038/s41467-020-17334-x

Figure Lengend Snippet: Amino acid preference surrounding pSer/pThr for PP1 and PP2A on the protein level from the phosphoproteomic data (Supplementary Data ). Amino acids with <25 raw counts in a given position were excluded and are marked in gray. a Color coding represents fold-change of the relative abundance of a given amino acid in a given position between phosphatase-sensitive/insensitive phosphorylation sites. b Direct comparison of PP1c and PP2Ac fold changes displayed in ( a ). Blue highlights amino acids selectively preferred by PP1c, and residues statistically different between PP1c and PP2Ac (adjusted p -value <0.01) according to a two-sided Fisher’s exact test are highlighted in bold. Please see Methods and Source Data for details and exact p -values. Source data are provided as a Source Data file.

Article Snippet: After three washes (5 min), membranes were then incubated with the following antibodies in TBS-T with 5% BSA overnight at 4 °C: α-GFP (1:2000, Abcam, ab6556), α-GAB2 (1:1000, CST, #3239), α-GAB2-pS210 (1:250, Symansis, customized purification), α-FLAG (1:1000, M2, Sigma-Aldrich, F3165), α-pan14-3-3 (1:500, SCBT, sc-629), α-PP1 (1:1000, SCBT, sc-7482), α-PP2A (1:1000, SCBT, sc-130237) or α-6His (1:2000, Abcam, ab9108).

Techniques: Phospho-proteomics, Comparison

Journal: Nature Communications

Article Title: Dissecting the sequence determinants for dephosphorylation by the catalytic subunits of phosphatases PP1 and PP2A

doi: 10.1038/s41467-020-17334-x

Figure Lengend Snippet: a Workflow of GFP-14-3-3β IP and subsequent MS analysis to identify 14-3-3-interacting proteins regulated by PP1. b Volcano plot of pairwise comparison between untreated/PP1c-treated samples (two-tailed Student’s t test, FDR 0.05). Proteins labeled in blue showed significant dissociation from 14-3-3 upon PP1c treatment and in orange represent SRSF proteins also identified in Table , 14–3–3 isoforms, and PP1 (added as recombinant protein). c Venn diagram showing overlap between the 56 proteins significantly dissociating from 14-3-3 upon PP1c treatment (but not binding GFP alone) compared to significantly dephosphorylated proteins (1967 p-sites on 1191 proteins) found in the PP1 phosphoproteomic experiments (t-test untreated vs. PP1 treated, see also Supplementary Fig. and Supplementary Data , ).

Article Snippet: After three washes (5 min), membranes were then incubated with the following antibodies in TBS-T with 5% BSA overnight at 4 °C: α-GFP (1:2000, Abcam, ab6556), α-GAB2 (1:1000, CST, #3239), α-GAB2-pS210 (1:250, Symansis, customized purification), α-FLAG (1:1000, M2, Sigma-Aldrich, F3165), α-pan14-3-3 (1:500, SCBT, sc-629), α-PP1 (1:1000, SCBT, sc-7482), α-PP2A (1:1000, SCBT, sc-130237) or α-6His (1:2000, Abcam, ab9108).

Techniques: Comparison, Two Tailed Test, Labeling, Recombinant, Binding Assay

Journal: Nature Communications

Article Title: Dissecting the sequence determinants for dephosphorylation by the catalytic subunits of phosphatases PP1 and PP2A

doi: 10.1038/s41467-020-17334-x

Figure Lengend Snippet: a The acidic groove is much more pronounced in PP1c compared to PP2Ac. Structures for PP1 catalytic subunit alpha isoform (PDB ID 3EGG, chain A) and PP2A catalytic subunit alpha isoform (PDB ID 4I5L, chain C) were retrieved from www.pdb.org (accessed 25 Oct 2019) and inspected in PyMOL v2.3.3. * marks the catalytic cleft containing two Mn 2+ ions. Color coding between −5 (red) to +5 (blue) K b T/e . b Aligned structures of PP1c and PP2Ac (see above for details) highlighting residues which determine differences in acidic properties (orange spheres). The sequence alignment to determine corresponding residues was carried out in Needle (EMBOSS). Color coding of structures: PP1c in blue/black, PP2Ac in red/gray. Residues constituting the acidic groove have previously been defined by Zhang et al. c Interaction of PP2Ac with B56/B´/PR61 based on the crystal structure of the trimeric holoenzyme with the scaffolding subunit A (PR65) derived from PDB ID 2NPP . Negatively charged amino acids within the binding regions of B56 to PP2Ac are highlighted. d The holoenzyme of PP1 has an intrinsic preference for basic motifs due to the composition of the acidic groove of the catalytic core protein. PP2A holoenzymes are per se not primed towards amino acid sequence features of 14-3-3 or PKA motifs, but need to associate to regulatory subunits such as B56 carrying acidic patches to achieve basophilic sequence specificity. e Both, PP1 and PP2A holoenzymes have a preference for pT due to a higher catalytic efficiency of their respective catalytic subunits towards pT over pS.

Article Snippet: After three washes (5 min), membranes were then incubated with the following antibodies in TBS-T with 5% BSA overnight at 4 °C: α-GFP (1:2000, Abcam, ab6556), α-GAB2 (1:1000, CST, #3239), α-GAB2-pS210 (1:250, Symansis, customized purification), α-FLAG (1:1000, M2, Sigma-Aldrich, F3165), α-pan14-3-3 (1:500, SCBT, sc-629), α-PP1 (1:1000, SCBT, sc-7482), α-PP2A (1:1000, SCBT, sc-130237) or α-6His (1:2000, Abcam, ab9108).

Techniques: Sequencing, Scaffolding, Derivative Assay, Binding Assay