Journal: bioRxiv

Article Title: Improved in situ Characterization of Proteome-wide Protein Complex Dynamics with Thermal Proximity Co-Aggregation

doi: 10.1101/2023.02.13.528386

Figure Lengend Snippet: (a) Dynamics of CORUM-annotated protein complexes under glucose deprivation as profiled by Slim-TPCA. Complexes are grouped based on their functions. (b) K562 cells were incubated in presence and absence of glucose followed by quantification of ATP based on luminescence intensity (n = 3 independent biological experiments). Data are presented as mean values ± s.d. (error bars). (c) K562 cells were incubated in presence and absence of glucose, harvested at different time points and analyzed by immunoblottingusing Phospho-Rb (Ser780) antibody, Phospho-Histone H3(Ser10) antibody, and Phospho-CDK Substrate antibody. GAPDH antibody and β-Tubulin antibody were used as loading control.

Article Snippet: Phospho-Rb (Ser780) antibody, Phospho-Histone H3(Ser10) antibody, Phospho-CDK Substrate antibody, and β-Tubulin antibody were obtained from Cell Signaling Technology (Danvers, USA).

Techniques: Incubation

Journal: International Journal of Biological Sciences

Article Title: ERK-mediated Cytoplasmic Retention of USP11 Contributes to Breast Cancer Cell Proliferation by Stabilizing Cytoplasmic p21

doi: 10.7150/ijbs.71327

Figure Lengend Snippet: USP11 is phosphorylated by activated ERK1/2 at serine 905. (A and B) MCF-7 cell lysates were immunoprecipitated using anti-USP11 (A) or anti-ERK1/2 antibody (B). The immunoprecipitates were then examined with indicated antibodies. (C) MCF-7 cells were treated with DMSO or U0126 (5 µM) for 6 h. Cell lysates were immunoprecipitated with anti-USP11 antibody, followed by western blotting with anti-p-Ser/Thr antibody and other indicated antibodies. (D) Sequence alignment of the ERK1/2 phosphorylation site within USP11 orthologs from different species. (E) MCF-7 cells were transfected with Flag-USP11 wt , Flag-USP11 S905A , empty vector plasmids for 24 h, and then treated with DMSO or U0126 (5 µM) for 6 h. Cell lysates were immunoprecipitated using anti-Flag antibody, followed by western blotting with anti-phospho-MAPK/CDK substrates antibody and other indicated antibodies.

Article Snippet: UltraSignal ECL (catalog no. 4AW011-100) was purchased from 4A Biotech Co., Ltd. Antibodies information: anti-USP11 (Santa Cruz, catalog no. sc-365528/ Abcam, catalog no. ab109232); anti-p21 (Santa Cruz, catalog no. sc-397/ Cell Signaling Technology, catalog no. 2947S); anti-Flag (MBL, catalog no. M185-3L); anti-Myc (MBL, catalog no. M192-3); anti-HA (MBL, catalog no. M180-3); anti-ERK1/2 (Cell Signaling Technology, catalog no. 4695S); anti-phospho-ERK1/2 (Cell Signaling Technology, catalog no. 4376S); anti-phospho-MAPK/CDK Substrates (Cell Signaling Technology, catalog no. 2325S); anti-phosphoserine/threonine (BD Transduction Laboratories, catalog no. M180-3); anti-GAPDH (COOLRUN Life Science, catalog no. AT0002); Dylight 488 (Thermo Fisher Scientific, catalog no. #35502); Dylight 594 (Thermo Fisher Scientific, catalog no. #35560).

Techniques: Immunoprecipitation, Western Blot, Sequencing, Transfection, Plasmid Preparation

Journal: PLoS ONE

Article Title: Kinase Activity Profiling of Pneumococcal Pneumonia

doi: 10.1371/journal.pone.0018519

Figure Lengend Snippet: Western blot (a) and quantification of all CDK phosphorylated substrates (b) revealed a decrease in CDK activity. Quantification was performed for all positive signals on the blot and corrected for β-actin. Data are represented as mean ± SD of n = 3.

Article Snippet: Blots were done for: AMPK-α/pAMPK-α, GSK-3β/pGSK-3β, pan-CDK phospho-substrate, β-catenin (all antibodies from Cellsignaling Technology, Boston, MA, USA) and β-actin (Santacruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Western Blot, Activity Assay

Journal: PLoS ONE

Article Title: Phosphorylation Regulates SIRT1 Function

doi: 10.1371/journal.pone.0004020

Figure Lengend Snippet: A. Affinity-purified SIRT1 is phosphorylated, and can be dephosphorylated by λppase. Top panel, western blot of total SIRT1; middle panel, western blot of SIRT1 phosphorylated at the Cdk consensus site; bottom panel, ProQ-diamond stained blot showing total phosphorylated SIRT1. Numbers above the lanes indicate the relative doses of λppase used to treat FLAG-SIRT1 for 1 hour. Overnight treatment (O/N) was done with a 1/10 dilution of λppase. B. Schematic of the phosphorylated residues in SIRT1 identified by mass spectrometry, as shown in . P, phosphorylated residue. C. 3D structure prediction of full-length human SIRT1 with the positions of phosphorylated residues identified by mass spectrometry (red symbols). Amino terminal domain, green; catalytic core domain, blue; carboxy terminal domain, yellow. The Rossmann fold supporting the catalytic groove in the core domain is shown in red. The two mutated residues described in the text (Thr530 and Ser540) are indicated with asterisks.

Article Snippet: The following antibodies were used: Peroxidase-conjugated AffiniPure Goat-anti-Mouse IgG and biotinylated anti-Mouse light chain (Jackson ImmunoResearch, West Grove, PA, #115-035-100, 1∶10,000), Peroxidase-conjugated AffiniPure Goat anti-Rabbit IgG (Jackson ImmunoResearch #110-035-144, 1∶10,000), anti-Cdc2 (Cdk1) mAb (Santa Cruz Biotechnology, Santa Cruz, CA, sc-8395, 1∶1,000), anti-Cdk2 pAb (Abcam, Cambridge, MA, ab7954, 1∶1,000), anti-Cdk4 pAb (Abcam ab2945, 1∶1,000), anti-Cdk6 mAb (Abcam ab3126, 1∶1,000), anti-cyclinA pAb (Abcam ab7956, 1∶1,000), anti-cyclinB1 pAb (Abcam ab7957, 1∶1,000), anti-FLAG M2 mAb (Sigma-Aldrich F3165, 1∶2,000), anti-SIRT1 N-terminal polyclonal antibody (Millipore #07-131, 1∶1,000), anti-SIRT1 C-terminal polyclonal antibody AS-16 (Sigma-Aldrich #S5313, 1∶1,000), anti-SIRT1 monoclonal antibody 2G1/F7 (Millipore #05-707, 1∶1,000), anti-phospho serine Cdk substrate antibody (Cell Signaling Technology, Danvers, MA, #2324, 1∶2,000), anti-GAPDH antibody (Abcam ab8245, 1∶5,000), and anti-pericentrin antibody (Covance, Richmond, CA, PRB-432C, 1∶300).

Techniques: Affinity Purification, Western Blot, Staining, Mass Spectrometry

Journal: PLoS ONE

Article Title: Phosphorylation Regulates SIRT1 Function

doi: 10.1371/journal.pone.0004020

Figure Lengend Snippet: Conservation of phosphorylated residues, cyclin recognition motifs, and Cdk substrate motifs among SIRT1 orthologs of 12 different species.

Article Snippet: The following antibodies were used: Peroxidase-conjugated AffiniPure Goat-anti-Mouse IgG and biotinylated anti-Mouse light chain (Jackson ImmunoResearch, West Grove, PA, #115-035-100, 1∶10,000), Peroxidase-conjugated AffiniPure Goat anti-Rabbit IgG (Jackson ImmunoResearch #110-035-144, 1∶10,000), anti-Cdc2 (Cdk1) mAb (Santa Cruz Biotechnology, Santa Cruz, CA, sc-8395, 1∶1,000), anti-Cdk2 pAb (Abcam, Cambridge, MA, ab7954, 1∶1,000), anti-Cdk4 pAb (Abcam ab2945, 1∶1,000), anti-Cdk6 mAb (Abcam ab3126, 1∶1,000), anti-cyclinA pAb (Abcam ab7956, 1∶1,000), anti-cyclinB1 pAb (Abcam ab7957, 1∶1,000), anti-FLAG M2 mAb (Sigma-Aldrich F3165, 1∶2,000), anti-SIRT1 N-terminal polyclonal antibody (Millipore #07-131, 1∶1,000), anti-SIRT1 C-terminal polyclonal antibody AS-16 (Sigma-Aldrich #S5313, 1∶1,000), anti-SIRT1 monoclonal antibody 2G1/F7 (Millipore #05-707, 1∶1,000), anti-phospho serine Cdk substrate antibody (Cell Signaling Technology, Danvers, MA, #2324, 1∶2,000), anti-GAPDH antibody (Abcam ab8245, 1∶5,000), and anti-pericentrin antibody (Covance, Richmond, CA, PRB-432C, 1∶300).

Techniques:

Journal: PLoS ONE

Article Title: Phosphorylation Regulates SIRT1 Function

doi: 10.1371/journal.pone.0004020

Figure Lengend Snippet: A/B. NAD + -dependent deacetylase activity of SIRT1 in the presence of CIP (A) or λppase (B) with or without phosphatase inhibitors. Western blots below each bar graph indicate the amount of SIRT1 in the reaction (top panel) and the degree of SIRT1 phosphorylation represented by Cdk-pSer signal (bottom panel). C. Deacetylase activity of affinity-purified FLAG-SIRT1 in the presence (left) or absence (right) of NAD + . Blue bars, no nicotinamide added; red bars, + nicotinamide. *, p<0.005; **, p<0.02; ***, p<0.002; #, p<0.01; ##, p<0.001 (Student's t -test). Error bars indicate +/−SEM.

Article Snippet: The following antibodies were used: Peroxidase-conjugated AffiniPure Goat-anti-Mouse IgG and biotinylated anti-Mouse light chain (Jackson ImmunoResearch, West Grove, PA, #115-035-100, 1∶10,000), Peroxidase-conjugated AffiniPure Goat anti-Rabbit IgG (Jackson ImmunoResearch #110-035-144, 1∶10,000), anti-Cdc2 (Cdk1) mAb (Santa Cruz Biotechnology, Santa Cruz, CA, sc-8395, 1∶1,000), anti-Cdk2 pAb (Abcam, Cambridge, MA, ab7954, 1∶1,000), anti-Cdk4 pAb (Abcam ab2945, 1∶1,000), anti-Cdk6 mAb (Abcam ab3126, 1∶1,000), anti-cyclinA pAb (Abcam ab7956, 1∶1,000), anti-cyclinB1 pAb (Abcam ab7957, 1∶1,000), anti-FLAG M2 mAb (Sigma-Aldrich F3165, 1∶2,000), anti-SIRT1 N-terminal polyclonal antibody (Millipore #07-131, 1∶1,000), anti-SIRT1 C-terminal polyclonal antibody AS-16 (Sigma-Aldrich #S5313, 1∶1,000), anti-SIRT1 monoclonal antibody 2G1/F7 (Millipore #05-707, 1∶1,000), anti-phospho serine Cdk substrate antibody (Cell Signaling Technology, Danvers, MA, #2324, 1∶2,000), anti-GAPDH antibody (Abcam ab8245, 1∶5,000), and anti-pericentrin antibody (Covance, Richmond, CA, PRB-432C, 1∶300).

Techniques: Histone Deacetylase Assay, Activity Assay, Western Blot, Affinity Purification

Journal: PLoS ONE

Article Title: Phosphorylation Regulates SIRT1 Function

doi: 10.1371/journal.pone.0004020

Figure Lengend Snippet: A. Western blots of Cdk-phosphorylated (top panel) and total SIRT1 (bottom panel) following treatment with increasing doses of lambda phosphatase (λppase). The upper band corresponds to the full-length SIRT1. B. Levels of SIRT1 phosphorylation at Cdk-specific sites in extracts used as substrates for in vitro kinase assays shown in C. C. Autoradiographs (top panels) and Coomassie blue-stained gels (bottom panels) of SIRT1 and histone H1 phosphorylated by the kinases indicated on left. 0, 0.01, 0.1, 1, relative doses of λppase used; %P, relative percentage of Cdk phospho-serine signal compared to control.

Article Snippet: The following antibodies were used: Peroxidase-conjugated AffiniPure Goat-anti-Mouse IgG and biotinylated anti-Mouse light chain (Jackson ImmunoResearch, West Grove, PA, #115-035-100, 1∶10,000), Peroxidase-conjugated AffiniPure Goat anti-Rabbit IgG (Jackson ImmunoResearch #110-035-144, 1∶10,000), anti-Cdc2 (Cdk1) mAb (Santa Cruz Biotechnology, Santa Cruz, CA, sc-8395, 1∶1,000), anti-Cdk2 pAb (Abcam, Cambridge, MA, ab7954, 1∶1,000), anti-Cdk4 pAb (Abcam ab2945, 1∶1,000), anti-Cdk6 mAb (Abcam ab3126, 1∶1,000), anti-cyclinA pAb (Abcam ab7956, 1∶1,000), anti-cyclinB1 pAb (Abcam ab7957, 1∶1,000), anti-FLAG M2 mAb (Sigma-Aldrich F3165, 1∶2,000), anti-SIRT1 N-terminal polyclonal antibody (Millipore #07-131, 1∶1,000), anti-SIRT1 C-terminal polyclonal antibody AS-16 (Sigma-Aldrich #S5313, 1∶1,000), anti-SIRT1 monoclonal antibody 2G1/F7 (Millipore #05-707, 1∶1,000), anti-phospho serine Cdk substrate antibody (Cell Signaling Technology, Danvers, MA, #2324, 1∶2,000), anti-GAPDH antibody (Abcam ab8245, 1∶5,000), and anti-pericentrin antibody (Covance, Richmond, CA, PRB-432C, 1∶300).

Techniques: Western Blot, In Vitro, Staining