Journal: bioRxiv

Article Title: Ionizable networks mediate pH-dependent allostery in SH2 signaling proteins

doi: 10.1101/2024.08.21.608875

Figure Lengend Snippet: (a) Schematic for in silico pK a prediction method for proteins with solved structures (see text and methods for details). Briefly, all available structures in the protein database are curated, electrostatic properties are calculated using PROPKA, results are filtered for ionizable residues with physiologically relevant predicted pK a values, and data are visualized in a 3D structure or a 2D residue interaction network. (b) Crystal structure of SHP2 shown in cartoon and surface format (PDB ID:2SHP). Protein tyrosine phosphatase (PTP) domain colored in grey, SH2 domains colored in yellow. (c) Structure of SHP2 (PDB ID:2SHP) with protein tyrosine phosphatase (PTP) domain in grey and SH2 domains in yellow. Residues identified through in silico ionizable network prediction pipeline shown in spheres. Residues with predicted pK a shifts (cyan) cluster with ionizable interactors (magenta) across the phosphatase-SH2 domain interaction interface of SHP2. (d) Table of predicted pK a s for cyan residues identified using in silico ionizable network prediction pipeline on 47 SHP2 structures (mean ± SD). (e) Residue interaction network of residues with predicted pK a shifts (cyan) and their ionizable interactors (magenta). Length of edges reflect the strength of the coulombic interaction, with stronger coulombic interactions having shorter edge lengths (f) Zoom of SHP2 structure at the PTP-SH2 interaction interface. Networked residues from a and b are shown in stick. Residues with predicted pK a shifts in cyan and ionizing interactors in magenta.

Article Snippet: Full-length SHP2 variants were cloned into the pGEX-4TI SHP2 WT plasmid , pGEX-4T1 SHP2 WT was a gift from Ben Neel (Addgene plasmid # 8322 ; http://n2t.net/addgene:8322 ; RRID:Addgene_8322)) and expressed in E. Coli for purification.

Techniques: In Silico, Residue

Journal: bioRxiv

Article Title: Ionizable networks mediate pH-dependent allostery in SH2 signaling proteins

doi: 10.1101/2024.08.21.608875

Figure Lengend Snippet: (a) Wild-type (WT) SHP2 in vitro phosphatase activity curves with increasing concentrations of generic substrate p-Nitrophenyl Phosphate (PNPP) at buffer pH ranging from 6.1 to 8.0. (mean ± SEM; N=3 from ≥2 different protein preparations) (b) Double mutant (H116A/E252A) SHP2 in vitro phosphatase activity, assays performed as in A. (mean ± SEM; N=3 from ≥2 different protein preparations) (c) Plot of k cat vs. pH for WT and double mutant (H116A/E252A) SHP2 activity. Calculated from activity curves in a and b. (mean ± SEM) (d) Single-mutant H116A-SHP2 in vitro phosphatase activity, assays performed as in a. (mean ± SEM; N=3 from ≥2 different protein preparations) (e) Single-mutant E252A-SHP2 in vitro phosphatase activity, assays performed as in a. (mean ± SEM; N=3, from ≥2 different protein preparations) (f) Plot of K cat vs. pH for WT and double mutant (H116A/E252A) SHP2 activity. Calculated from activity curves in a, d and e. (mean ± SEM) (g) Proposed pH-sensing mechanism where SH2 domain (yellow) binding to catalytic domain (grey) is titratable by pH. (h) CpHMD (see methods for details) was performed on SHP2 at pH values from 4.0-10.0 (see supplemental videos). Shown are overlapping views of SHP2 structures at the start of CpHMD simulation (t = 0 ns) and end of simulation (t = 8 ns) for pH values 4.5 (pink), 5.5 (purple), 6.5 (blue) and 8.5 (yellow).

Article Snippet: Full-length SHP2 variants were cloned into the pGEX-4TI SHP2 WT plasmid , pGEX-4T1 SHP2 WT was a gift from Ben Neel (Addgene plasmid # 8322 ; http://n2t.net/addgene:8322 ; RRID:Addgene_8322)) and expressed in E. Coli for purification.

Techniques: In Vitro, Activity Assay, Mutagenesis, Binding Assay

Journal: bioRxiv

Article Title: Ionizable networks mediate pH-dependent allostery in SH2 signaling proteins

doi: 10.1101/2024.08.21.608875

Figure Lengend Snippet: (a) pHi measurements of MCF10A cells. Cells were treated with 25 μM EIPA + 30 μM S0858 for 1 hour to lower pH to 7.10. To raise pHi, cells were treated with 30 mM ammonium chloride for 1 hour to raise pH to 8.00. Untreated cells had a pHi of 7.45. Scatter plot shows (median ± interquartile range, N =10) (b) Representative immunoblots of SHP2, Gab1, and phospho-SHP2 (pY542) from SHP2 immune complexes (SHP2 IP) or whole cell lysates (Cell Lysate) isolated from MCF10A cells prepared as in A. (c) Quantification of replicate data collected as in B. Data was normalized to control in each biological replicate. Scatter plot shows (mean ± SEM, N=4). I (d) Quantification of Co-IP of SHP2 shown in b. Immunoblot intensities in the treatment conditions were normalized to control in each biological replicate. Scatter plot shows (mean ± SEM, N=7). (e) Representative images of MCF10A cells expressing the SHP2 activity reporter (Grb2 TagBFP) pseudocolored on an intensiometric scale. Images show cells prior to manipulating pHi with nigericin buffer (Pre Nigericin) (see methods for details), 50s - after manipulating pHi, and 900s after manipulating pHi. Scale bars: 25μm (f) Quantification of images as in E. Membrane intensity of SHP2 activity reporter was photobleach-corrected and then normalized to initial intensity over time. Line trace shows from single-cell data (mean ± SEM) (6.7 pH, n=30, 7.4 pH, n=30, 7.8 pH, n=25, control, n=28) collected across N=3 biological replicates. (g) Quantification of endpoint membrane intensities of single cells collected as described in f. Scatter plot shows (median ± interquartile range, N = 5) (h) Representative immunoblot of lysates prepared from MCF10A cells expressing either WT SHP2 or H116A/E252A SHP2 and treated as described in a. Immunoblots show total and pY542-SHP2 under low, control, and high pHi conditions. (i) Quantification of replicate data collected as in h. Scatter plots show (mean ± SEM, N=3). Intensities were normalized to the corresponding control condition. For a and g significance was determined using the Kruskal-Wallis test. For c, d, and i significance was determined using a ratio paired t-test to compare between treatment conditions and a one-sample t-test to compare to control. * p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Article Snippet: Full-length SHP2 variants were cloned into the pGEX-4TI SHP2 WT plasmid , pGEX-4T1 SHP2 WT was a gift from Ben Neel (Addgene plasmid # 8322 ; http://n2t.net/addgene:8322 ; RRID:Addgene_8322)) and expressed in E. Coli for purification.

Techniques: Western Blot, Isolation, Control, Co-Immunoprecipitation Assay, Expressing, Activity Assay, Membrane

Journal: bioRxiv

Article Title: Ionizable networks mediate pH-dependent allostery in SH2 signaling proteins

doi: 10.1101/2024.08.21.608875

Figure Lengend Snippet: (a) Schematic of pH-driven activation and inhibition of SHP2. At low pH, the SH2 domain is unbound and SHP2 becomes signaling active with increased phosphorylation of Y542, increased GAB1 binding, and increased Grb2 recruitment. (b) Schematic of pH-driven activation and inhibition of Src. At low pH, the SH2 domain is unbound and c-Src becomes signaling active with increased phosphorylation of Y416, decreased phosphorylation of Y527, and increased membrane recruitment.

Article Snippet: Full-length SHP2 variants were cloned into the pGEX-4TI SHP2 WT plasmid , pGEX-4T1 SHP2 WT was a gift from Ben Neel (Addgene plasmid # 8322 ; http://n2t.net/addgene:8322 ; RRID:Addgene_8322)) and expressed in E. Coli for purification.

Techniques: Activation Assay, Inhibition, Phospho-proteomics, Binding Assay, Membrane

Journal: bioRxiv

Article Title: Mitotic Regulators and the SHP2-MAPK Pathway Promote Insulin Receptor Endocytosis and Feedback Regulation of Insulin Signaling

doi: 10.1101/419911

Figure Lengend Snippet: IRS1/2 are required for insulin-activated IR endocytosis. a HepG2 cells stably expressing IR-GFP WT were transfected with the indicated siRNAs or siRNA-resistant Myc-IRS1, serum starved, treated without or with 100 nM insulin for 5 min, and stained with anti-GFP antibodies. b Quantification of the ratios of PM and IC IR-GFP signals of cells in ( a ) (mean ± SD; *p<0.0001). c Domains and YXXΦ motifs of human IRS1 and mouse IRS2. PH, pleckstrin homology domain; PTB, phosphotyrosine-binding domain. AP2M1- and SHP2-binding regions are indicated. YXXΦ motifs and phosphotyrosine sites of IR for SHP2 binding are shown as blue and red bars, respectively. The MAPK phosphorylation sites are labeled as green letters in the sequences. d 293FT cells stably expressing IR-GFP WT were transfected with the indicated siRNAs or siRNA-resistant Myc-IRS1, serum starved, treated without or with 100 nM insulin for 5 min, and stained with anti-GFP (IR; green), anti-Myc (IRS1; red), and DAPI (blue). (3YA, Y612A/Y632A/Y662A; 3YF, Y612F/Y632F/Y662F; 3SA, S616A/S636A/S666A; 3SD, S616D/S636D/S666D; Y2A, Y1179A/Y1229A). e Quantification of the ratios of PM and IC IR-GFP signals of cells in ( d ) (mean ± SD; *p<0.0001). f 293FT cells were serum starved and treated without or with 100 nM insulin for 5 min. Total cell lysate (TCL), anti-IRS1 IP, and IgG IP were blotted with anti-IRS1 and anti-AP2B1 antibodies. g Serum-starved primary hepatocytes were treated with DMSO or 10 µM SHP099 for 2 h and treated with 100 nM insulin for 5 min. Total cell lysate (TCL), anti-IRS1 IP were blotted with anti-IRS1 and anti-AP2B1 antibodies.

Article Snippet: Active SHP2 (2.9 µM, SignalChem) diluted in the phosphatase dilution buffer [50 mM imidazole, pH 7.2, 0.2% 2-mercaptoethanol, 65 ng/µl BSA] was incubated with IRS1 peptides (2.6 mM) at 37°C for the indicated time points.

Techniques: Stable Transfection, Expressing, Transfection, Staining, Binding Assay, Labeling

Journal: bioRxiv

Article Title: Mitotic Regulators and the SHP2-MAPK Pathway Promote Insulin Receptor Endocytosis and Feedback Regulation of Insulin Signaling

doi: 10.1101/419911

Figure Lengend Snippet: IRS1 promotes IR endocytosis and interacts with AP2. a Western blot analysis of cell lysates in . Asterisks indicate non-specific bands. b Domains and YXXΦ motifs of human IRS1. PH, pleckstrin homology domain; PTB, phosphotyrosine-binding domain. IRS1 fragments that can or cannot bind to AP2M1 are presented as red or black lines, respectively. YXXΦ motifs and phosphotyrosine sites for SHP2 binding are presented as blue and red bars, respectively. c Binding of IRS1 WT and mutants to GST or GST-AP2M1. Input and protein bound to beads were blotted with anti-Myc (IRS1) antibodies and stained with Coomassie (CBB). The relative band intensities are shown below (mean ± SD; n=3 independent experiments). d Binding of IRS1 WT and truncation mutants to GST or GST-AP2M1. Input and protein bound to beads were blotted with the indicated antibodies. The relative band intensities are shown below (n=2 independent experiments). e Sequence alignment of a conserved region in IRS1/2. Three YXXΦ motifs are boxed with red dashed lines. The phosphorylation sites of IR and MAPK are indicated as red and blue dots, respectively.

Article Snippet: Active SHP2 (2.9 µM, SignalChem) diluted in the phosphatase dilution buffer [50 mM imidazole, pH 7.2, 0.2% 2-mercaptoethanol, 65 ng/µl BSA] was incubated with IRS1 peptides (2.6 mM) at 37°C for the indicated time points.

Techniques: Western Blot, Binding Assay, Staining, Sequencing

Journal: bioRxiv

Article Title: Mitotic Regulators and the SHP2-MAPK Pathway Promote Insulin Receptor Endocytosis and Feedback Regulation of Insulin Signaling

doi: 10.1101/419911

Figure Lengend Snippet: The SHP2-MAPK pathway promotes insulin-activated IR endocytosis. a HepG2 cells expressing IR-GFP WT were starved, treated with the indicated inhibitors for 2 h, treated without or with 100 nM insulin for 20 min, and stained with anti-GFP (IR; green) and DAPI (blue). b Quantification of the ratios of PM and IC IR-GFP signals of cells in (A) (mean ± SD; *p<0.0001). c Binding of IRS1 peptides to AP2M1 (residues 160-435). Input and proteins bound to IRS1-peptide beads were analyzed by SDS-PAGE and stained with Coomassie (CBB). The relative band intensities are shown below (mean ± SD; n=4 independent experiments). d Isothermal titration calorimetry (ITC) analysis of binding between IRS1 peptides and AP2M1 (residue 160-435), with K d indicated. e The IRS1 peptides were incubated with active SHP2 for the indicated durations, spotted onto membranes, and detected with the anti-pY612-IRS1 antibody. f Quantification of the relative SHP2 activity in ( e ) (mean ± SD; n=4 independent experiments; *p<0.0001). g Model of the regulation of insulin-activated IR endocytosis by a phosphorylation switch on IRS1/2. Insulin-bound IR phosphorylates itself and IRS1/2, and activates the PI3K-AKT and MAPK pathways. SHP2 acts upstream of RAS-RAF and promotes the activation of MAPK pathway. p31 comet binds to the IR-bound MAD2 and blocks IR-AP2 association to prevent premature IR endocytosis. In feedback regulation, activated ERK1/2 phosphorylate S616 and other sites on IRS1. SHP2 binds to the C-terminal phospho-tyrosine site on IRS1 and dephosphorylates pY612 of the doubly phosphorylated IRS1 (pY612/pS616), thus promoting IRS1-AP2M1 association. p31 comet is released from MAD2 by an unknown mechanism, allowing the assembly of an MCC-like complex on IR. MAD2- and IRS1/2-dependent AP2 recruitment and clustering trigger clathrin-mediated IR endocytosis. h Ribbon diagram of the crystal structure of AP2M1 (residues 160-435) bound to pS-IRS1. pS-IRS1 is shown as sticks. i Surface drawing of AP2M1, with pS-IRS1 shown as sticks. j A close-up view of the surface drawing of AP2M1 colored by its electrostatic potential (blue, positive; red, negative; white, neutral). pS-IRS1 is shown as sticks.

Article Snippet: Active SHP2 (2.9 µM, SignalChem) diluted in the phosphatase dilution buffer [50 mM imidazole, pH 7.2, 0.2% 2-mercaptoethanol, 65 ng/µl BSA] was incubated with IRS1 peptides (2.6 mM) at 37°C for the indicated time points.

Techniques: Expressing, Staining, Binding Assay, SDS Page, Isothermal Titration Calorimetry, Incubation, Activity Assay, Activation Assay

Journal: bioRxiv

Article Title: Mitotic Regulators and the SHP2-MAPK Pathway Promote Insulin Receptor Endocytosis and Feedback Regulation of Insulin Signaling

doi: 10.1101/419911

Figure Lengend Snippet: SHP2 inhibition delays IR endocytosis and improves insulin sensitivity in mice. a , b Glucose tolerance test ( a ) and insulin tolerance test ( b ) of male mice fed HFD for 5 weeks. The mice were administered vehicle or SHP099 for 6 days. At 1 day after the last drug administration, experiments were performed. Vehicle, n=12; SHP099, n=10; mean ± SEM. c Body weight of mice administered vehicle or SHP099 at 7 days post administration. Mean ± SD. d HFD-fed mice were administered vehicle or SHP099 for 5 days. The mice were fasted overnight and administered vehicle or SHP099 once more. At 2 h after the last administration, the mice were injected with or without 1 U insulin via inferior vena cava. The livers were collected at the indicated time points and the sections were stained with anti-IR (red) and DAPI (blue). Scale bars, 5 µm. e Quantification of the ratios of plasma membrane (PM) and intracellular compartments (IC) IR signals of the livers in ( d ) (mean ± SD; *p<0.0001). f - h The levels of fasting serum insulin ( f ) and C-peptide ( g ), and the ratio of C-peptide:insulin ( h ) in mice fed normal chow or HFD for 5 weeks. The mice were administered vehicle or SHP099 for 6 days. i HepG2 cells stably expressing IR-GFP were transfected with CEACAM1 siRNAs, serum starved, treated without or with 100 nM insulin form 5 min, and stained with anti-GFP and DAPI. Quantification of the ratios of PM and IC IR-GFP signals of cells was shown (mean ± SD). j Western blot analysis of cell lysates in ( i ). k Model of the regulation of insulin-activated IR endocytosis by CEACAM1, the MAD2–CDC20– BUBR1 module, and the SHP2-IRS1/2 module.

Article Snippet: Active SHP2 (2.9 µM, SignalChem) diluted in the phosphatase dilution buffer [50 mM imidazole, pH 7.2, 0.2% 2-mercaptoethanol, 65 ng/µl BSA] was incubated with IRS1 peptides (2.6 mM) at 37°C for the indicated time points.

Techniques: Inhibition, Injection, Staining, Stable Transfection, Expressing, Transfection, Western Blot

Journal: bioRxiv

Article Title: Mitotic Regulators and the SHP2-MAPK Pathway Promote Insulin Receptor Endocytosis and Feedback Regulation of Insulin Signaling

doi: 10.1101/419911

Figure Lengend Snippet: Depletion of SHP2 by shRNA delays IR endocytosis and improves insulin sensitivity in mice. a The level of SHP2 in liver, skeletal muscle and epididymal WAT from mice fed HFD for 5 weeks. The mice were injected with AAV-control (Ctrl) or SHP2 shRNA. At 17 days after injection, the mice were fasted overnight and injected with or without 1 U insulin via inferior vena cava. The livers were collected at the indicated time points. WAT and skeletal muscle were collected at 2 min and 3 min after the indicated time points, respectively. Lysates were prepared from these tissues and subjected to quantitative immunoblotting with the indicated antibodies. b , c Glucose tolerance test ( b ) and insulin tolerance test ( c ) in mice injected with AAV-Ctrl or AAV-SHP2 shRNA and fed HFD. Experiments were performed at 2 weeks after injection. n=6; mean ± SEM. d Body weight in HFD-fed mice injected with AAV-Ctrl or AAV-SHP2 shRNA. Mean ± SD. e HFD-fed mice were injected with AAV-Ctrl or AAV-SHP2. At 17 days after injection, the mice were fasted overnight and injected with or without 1U insulin via inferior vena cava. The livers were collected at the indicated time points and the sections were stained with anti-IR (red) and DAPI (blue). Scale bars, 5 µm. f Quantification of the ratios of PM and IC IR signals of the livers in ( e ) (mean ± SD; *p<0.0001).

Article Snippet: Active SHP2 (2.9 µM, SignalChem) diluted in the phosphatase dilution buffer [50 mM imidazole, pH 7.2, 0.2% 2-mercaptoethanol, 65 ng/µl BSA] was incubated with IRS1 peptides (2.6 mM) at 37°C for the indicated time points.

Techniques: shRNA, Injection, Western Blot, Staining

Journal: bioRxiv

Article Title: Mitotic Regulators and the SHP2-MAPK Pathway Promote Insulin Receptor Endocytosis and Feedback Regulation of Insulin Signaling

doi: 10.1101/419911

Figure Lengend Snippet: Mitotic regulators and SHP2 promote feedback inhibition of IR. a Insulin signaling in the liver from mice fed HFD for 5 weeks. The mice were administered vehicle or SHP099 for 5 days, fasted overnight, and administered vehicle or SHP099 once more. At 2 h after the last administration, the mice were injected with or without 1 U insulin via inferior vena cava. The livers were collected at the indicated time points. Lysates were prepared from these tissues and subjected to quantitative immunoblotting with the indicated antibodies. b Quantification of the blots in ( a ). Mean ± SD; *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. c Targeting feedback regulation of IR endocytosis for diabetes treatment. Left panel depicts the feedback regulation of IR endocytosis by ERK1/2 and SHP2 during unperturbed insulin signaling. Right panel illustrates the mechanism by which SHP2 inhibitor (SHP099) or shRNA blocks growth-promoting IR signaling and IR endocytosis, and prolongs insulin signaling through the PI3K-AKT pathway, which controls metabolism.

Article Snippet: Active SHP2 (2.9 µM, SignalChem) diluted in the phosphatase dilution buffer [50 mM imidazole, pH 7.2, 0.2% 2-mercaptoethanol, 65 ng/µl BSA] was incubated with IRS1 peptides (2.6 mM) at 37°C for the indicated time points.

Techniques: Inhibition, Injection, Western Blot, shRNA

Journal: PLoS Pathogens

Article Title: HTLV-1 bZIP Factor Enhances T-Cell Proliferation by Impeding the Suppressive Signaling of Co-inhibitory Receptors

doi: 10.1371/journal.ppat.1006120

Figure Lengend Snippet: (A) Phosphorylation levels of SHP-2 (Tyr580) in CD4 + T cells of non-Tg and HBZ-Tg mice were analyzed by flow cytometry. Splenocytes of non-Tg or HBZ-Tg mice (8 weeks old) were stained with anti-CD4 and pSHP-2 (Tyr580) antibodies. (B) The phosphorylation level of SHP-2 (Tyr580) of HBZ-transduced murine primary CD4 + T cells was analyzed by flow cytometry. After HBZ transduction, cells were stimulated with anti-CD3/PD-L1.Fc-coated beads at bead-to-cell ratio of 1:1 for 6 hours. (C-E) Dephosphorylation of ZAP-70, CD3ζ and PKCθ was analyzed by immunoblotting (left). Jurkat-mock and Jurkat-HBZ cells were stimulated with 20 mM H 2 O 2 or 0.5 mM pervanadate for 0, 5, 15, 30 or 45 min. Phosphorylation levels were also analyzed by densitometry (right). Relative protein levels were calculated as the ratio of phosphorylated protein to total protein.

Article Snippet: Anti-phospho-SHP-2 (Tyr580) antibody was from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Phospho-proteomics, Flow Cytometry, Staining, Transduction, De-Phosphorylation Assay, Western Blot

Journal: Biochemistry

Article Title: Targeted degradation of the oncogenic phosphatase SHP2

doi: 10.1021/acs.biochem.1c00377

Figure Lengend Snippet: (A) Chemical structure of RMC-4550 and X-ray crystal structure of SHP2 in complex with RMC-4550 (PDB code 7RCT). Surface representation of SHP2 in complex with RMC-4550 bound in the central tunnel formed at the interface of N-SH2 (green), C-SH2 (blue) and PTP (wheat) domains. (B) Chemical structures of RMC-4550-based PROTAC candidates, R1–1C, R1–3C and R1–5C.

Article Snippet: Antibodies used in this study were obtained commercially from the following sources: SHP2 (Bethyl, #A301–544A), Phospho-Thr202/Tyr204-Erk1/2 (CST, #9101), Cereblon (CST, #71810), b-actin (Millipore-Sigma, #A1978), b -tubulin (CST, #2146), GAPDH (CST, #5174).

Techniques:

Journal: Biochemistry

Article Title: Targeted degradation of the oncogenic phosphatase SHP2

doi: 10.1021/acs.biochem.1c00377

Figure Lengend Snippet: (A) Inhibition of SHP2-F285S- or PTP-mediated DIFMUP dephosphorylation by R1–1C, R1–3C, R1–5C and RMC-4550. MV4;11 cells were treated with increasing doses of R1–3C (B), R1–1C or R1–5C (C) for 24 h and subjected to Western blotting using SHP2, GAPDH and β-actin antibodies. Quantification of band intensities on the gels are shown below the blots.

Article Snippet: Antibodies used in this study were obtained commercially from the following sources: SHP2 (Bethyl, #A301–544A), Phospho-Thr202/Tyr204-Erk1/2 (CST, #9101), Cereblon (CST, #71810), b-actin (Millipore-Sigma, #A1978), b -tubulin (CST, #2146), GAPDH (CST, #5174).

Techniques: Inhibition, De-Phosphorylation Assay, Western Blot

Journal: Biochemistry

Article Title: Targeted degradation of the oncogenic phosphatase SHP2

doi: 10.1021/acs.biochem.1c00377

Figure Lengend Snippet: (A) Time course of SHP2 degradation by R1–5C (100 nM) in MV4;11 cells. Immunoblotting with SHP2 and β-actin antibodies. (B) CRBN−/− and parental MOLT4 cells were treated with increasing doses of R1–5C for 24 h and subjected to Western blotting using SHP2, CRBN and β-actin antibodies. Quantification of band intensities on the gels are shown below the blots.

Article Snippet: Antibodies used in this study were obtained commercially from the following sources: SHP2 (Bethyl, #A301–544A), Phospho-Thr202/Tyr204-Erk1/2 (CST, #9101), Cereblon (CST, #71810), b-actin (Millipore-Sigma, #A1978), b -tubulin (CST, #2146), GAPDH (CST, #5174).

Techniques: Western Blot

Journal: Biochemistry

Article Title: Targeted degradation of the oncogenic phosphatase SHP2

doi: 10.1021/acs.biochem.1c00377

Figure Lengend Snippet: (A-D) Scatterplots displaying relative fold-change in SHP2 abundance following treatment of MV4;11 cells with 100 nM R1–5C for 4 h (A), 8 h (B), 16 h (C) or 100 nM RMC-4550 (D). SHP2/PTPN11 is highlighted in red. Hits highlighted in blue in (C) and (D) indicate changes in abundance of proteins at 16 h time point due to secondary effects (such as transcriptional responses) of SHP2 degradation or inhibition. (E) Heatmap of the protein abundance changes in MV4;11 cells comparing treatment with 100 nM R1–1C (4 h and 16 h), 100 nM R1–3C (4 h and 16 h), 100 nM R1–5C (2 h, 4 h, 8 h and 16 h), 100 nM RMC-4550 (16 h) and 1 μM pomalidomide (5 h). The heatmap colors are scaled with red indicating a decrease in protein abundance (−2 log2 FC) and blue indicating an increase (2 log2 FC) in protein abundance.

Article Snippet: Antibodies used in this study were obtained commercially from the following sources: SHP2 (Bethyl, #A301–544A), Phospho-Thr202/Tyr204-Erk1/2 (CST, #9101), Cereblon (CST, #71810), b-actin (Millipore-Sigma, #A1978), b -tubulin (CST, #2146), GAPDH (CST, #5174).

Techniques: Inhibition