Journal: Nature

Article Title: Structure-function analysis of the SHOC2-MRAS-PP1C holophosphatase complex

doi: 10.1038/s41586-022-04928-2

Figure Lengend Snippet: Bio-layer interferometry analysis of SHOC2 ternary complex. Binding kinetic values for step-specific complex member binding (Interaction) are indicated. Association rate constant (k a ), dissociation rate constant (k d ), steady-state dissociation constant (K D ), and least squares fit (LSF) are shown. Values represent the mean of three replicates ± standard deviation. *Kinetics too irreversible to be determined.

Article Snippet: Beads were boiled in 2x laemmli buffer, and input/eluted IP samples were immunoblotted for V5 (CST 13202S), HA (CST 3724TS), and blotted for PP1CB (Thermo Scientific PA5-78117).

Techniques: Binding Assay, Standard Deviation

Journal: Nature

Article Title: Structure-function analysis of the SHOC2-MRAS-PP1C holophosphatase complex

doi: 10.1038/s41586-022-04928-2

Figure Lengend Snippet: a, Violin plot of SHOC2 mean positional viability for surface contacting residues between complex members, PP1C (green, n = 28 positions) and MRAS (maroon, n = 26 positions), compared to core-residues (brown, n = 198 positions) and surface non-contacting residues (yellow, n = 329). Center line represents median, whiskers represent the first and fourth quartiles, box edges represent the second and third quartiles. b, MIA PaCa-2 with knock-out of endogenous SHOC2 and stably re-expressing various SHOC2 gain-of-function (red) and loss-of-function (blue) variants were seeded in ultra-low attachment plates and cultured for 7 days. Viability endpoint via cell titer glow is presented on x-axis along scaled LFC from fitness screen with PaTu-8902. Error bars represent standard deviation of GILA CTG viability (n=6 technical replicates; representative of 3 biological replicates). Line (green) represent simple linear regression model, 95% confidence interval (black dashed lines), R2 (goodness of fit), and linear model p-value < 0.0001 (analysis of regression coefficient significantly non-zero) indicated. c, Wild type (WT) and gain- or loss-of-function (GOF/LOF) variants were stably expressed in KRAS mutant cell line MIA PaCa-2 with knock-out of endogenous SHOC2. d, Densitometry quantification of P-S259 RAF1 relative to total RAF1 and normalized to levels from wildtype expressing cells. Center line represents median and whiskers represent interquartile range. ***p<0.001, two-sided t-test between LOF (n = 5 variants) and GOF (n = 6 variants) SHOC2 alleles, representative of 3 biological replicates. e, Wild type (WT) and gain- or loss-of-function (GOF/LOF) variants were stably expressed in KRAS mutant cell line MIA PaCa-2 with knock-out of endogenous SHOC2. Cells were treated with the MEK1/2 inhibitor trametinib (10nM) for 24 hours prior to Western blot. f, Densitometry quantification of P-S259 RAF1 relative to total RAF1 and normalized to levels from wildtype expressing cells. Center line represents median and whiskers represent interquartile range. ***p<0.001, two-sided t-test between LOF (n = 6 variants) and GOF (n = 6 variants) SHOC2 alleles, representative of 3 biological replicates. g, Immunoprecipitation of V5-tagged SHOC2 variants in 293T cells co-transfected with HA-MRAS. h, Densitometry analysis of relative prey including endogenous PP1CB (yellow) and MRAS (maroon) normalized to V5 bait (y-axis) and DMS fitness score (LFC Z-score) (x-axis). Lines represent simple linear regression model, R2 (goodness of fit), and linear model p-value < 0.0001 (analysis of regression coefficient significantly non-zero) indicated, representatitve of 3 biological replicates. i, Deep mutational scanning results for N-terminal region of SHOC2 (residues 60-68) depicted via sequence logo plot per amino acid substitution at respective positions (ggseqlogo).

Article Snippet: Beads were boiled in 2x laemmli buffer, and input/eluted IP samples were immunoblotted for V5 (CST 13202S), HA (CST 3724TS), and blotted for PP1CB (Thermo Scientific PA5-78117).

Techniques: Inhibition, Knock-Out, Stable Transfection, Expressing, Cell Culture, Standard Deviation, Mutagenesis, Western Blot, Immunoprecipitation, Transfection, Sequencing

Journal: bioRxiv

Article Title: PCDH7 Promotes Cell Migration by Regulating Myosin Activity

doi: 10.1101/2021.09.21.460794

Figure Lengend Snippet: (A) Depiction of relevant clusters of PCDH7 isoform-c interactome determined by GFP mediated affinity pulldown followed by LC-MS/MS. Red inset depicts a set of phosphatase subunits and phosphatase regulatory proteins, green inset highlighting components of myosin complexes, and blue inset showing members of ERM proteins. (B) Immunostaining of RPE1 cells expressing PCDH7-GFP (green), MYPT1 (PPP1R12A) (red) and fluorescent phalloidin (blue). (C) Immunoblotting for GFP pulldown of EGFP-PP1CB-MYPT1 fusion, EGFP-PP1CB*-MYPT1 (D63A) mutant and control construct with cotransfection of PCDH7c-V5 construct. Membranes were blotted using anti-V5, anti-MYPT1, anti-GFP and anti-tubulin antibodies. I (Input), FT(Flow Through), E (Elution) (D) Quantification of proximity ligation assay (PLA) showing fluorescence dots per cell for PCDH7- GFP (anti-GFP) and MYPT1 (anti-MYPT1) interaction (n=29) along with single primary antibody controls, MYPT1 (n=30) and GFP (n=30). ****p<0.0001.

Article Snippet: For PCDH7- V5 constructs, PCDH7b and PCDH7c were cloned into PLEX_307 (Addgene#41392). eGFP- PP1CB-MYPT1 and eGFP-PP1CB(D63A)-MYPT1 were provided by Mathieu Bollen from KU, Leuven and generated as previously described ( ).

Techniques: Liquid Chromatography with Mass Spectroscopy, Immunostaining, Expressing, Western Blot, Mutagenesis, Construct, Cotransfection, Proximity Ligation Assay, Fluorescence

Journal: Antioxidants

Article Title: MnTE-2-PyP Suppresses Prostate Cancer Cell Growth via H 2 O 2 Production

doi: 10.3390/antiox9060490

Figure Lengend Snippet: Identification of MnTE-2-PyP-induced protein oxidation and PPPs activity measurements. ( A ) A representative image of Coomassie stained gels of non-oxidized proteins in PC3 cells. Proteins with reduced thiols were isolated by N-(biotinoyl)-N’-(iodoacetyl) ethylenediamine (BIAM) probe and selected regions of Coomassie gels were cut and sent for mass spectrometry analyses. ( B ) A representative image of a PP1CB western blot with ponceau staining in PC3 cells and corresponding densitometry analysis. ( C ) A representative image of a PP1CB western blot with ponceau staining in LNCaP cells and corresponding densitometry analysis. ( D ) A representative image of a PP1CB western blot with ponceau staining in PC3 and LNCaP cells and corresponding densitometry analysis. ( E ) PP1 activity measurement in PC3 cells with PBS or MnTE-2-PyP treatment. ( F ) Total PPP activity measurement in PC3 cells with PBS or MnTE-2-PyP treatment. ( G ) PP1 activity measurement in LNCaP cells with PBS or MnTE-2-PyP treatment. ( H ) Total PPP activity measurement in LNCaP cells with PBS or MnTE-2-PyP treatment. All data represent mean ± SD from at least three independent experiments. * p < 0.05 compared to PBS treatment.

Article Snippet: After blocking with 5% non-reduced fat milk in TBST for 1 h, the membranes were incubated overnight at 4 °C with the following primary antibodies: PP1CB (1:500), cyclin D1 (1:10,000), phospho-cyclin D1 (Thr 286, 1:1000), pRB, phospho-pRB (Ser780, 1:1000) (Cell Signaling Technology, Danvers, MA, USA) and p16 (1:5000), p21 (1:5000) (Abcam, Cambridge, MA, USA).

Techniques: Activity Assay, Staining, Isolation, Mass Spectrometry, Western Blot