Journal: Life Science Alliance

Article Title: α-Actinin-4 recruits Shp2 into focal adhesions to potentiate ROCK2 activation in podocytes

doi: 10.26508/lsa.202201557

Figure Lengend Snippet: MEFs seeded on FN-coated dishes were treated with or without FAK inhibitor 14 (15 μM) for 90 min for isolation of FAs as described in the Materials and Methods section. (A) Whole-cell lysate (WCL; 1%) and isolated FA fractions (FA) were subjected to Western analysis as indicated. Relative FAK Y397 phosphorylation and levels of Shp2 versus vinculin in FA fractions from four independent experiments were measured. (B) FA fractions were incubated with 50 μg of His-tagged Shp2 N-SH2 recombinant protein followed by pulled-down with Ni-beads, SDS–PAGE, and sliver staining. By mass spectrometry analysis, the band indicated by * was identified as α-actinin-4 and α-actinin-1. (C, D) FA fractions isolated from non-treated MEFs were immunoprecipitated with anti-Shp2 antibody followed by Western blotting analysis as indicated. The concentration of Shp2 and α-actinin-4 in the IP input sample might be too low to be detected here. (E) FA fractions were incubated with or without 400 U of λPPase for 30 min before pull-down with His-N-SH2 proteins and Ni-beads. The levels of pulled-down α-actinin-4 was detected by Western blotting as indicated. Data are mean ± SD. * P < 0.05, ** P < 0.01 (two-tailed, paired t test). Source data are available for this figure.

Article Snippet: Anti-paxillin antibody and mouse collagen type-IV from BD Biosciences; anti-β-actin, anti-β-tubulin, and anti-flag antibodies, FITC-phalloidin, ROCK inhibitor Y27632, and Shp2 inhibitor IIB-08 from Sigma-Aldrich; FAK inhibitor 14 from Tocris Bioscience, anti-pY397FAK, anti-pY31-paxillin, and anti-ZO-1 antibodies from Invitrogen; anti-pY542 Shp2, anti-pT18/pS19-MLC, and anti-MLC antibodies from Cell Signaling; anti-α-actinin-1 antibody from Chemicon; anti-α-actinin-4 and anti-phosphotyrosine (clone 4G10) antibodies from Millipore; anti-FAK, anti-Shp2, anti-ROCK1, and anti-ROCK2, antibodies from Santa Cruz Biotechnology Inc.; anti-synaptopodin antibody from Novus; anti-podocin antibody from Abcam; λ protein phosphatase from New England BioLabs.

Techniques: Isolation, Western Blot, Phospho-proteomics, Incubation, Recombinant, SDS Page, Staining, Mass Spectrometry, Immunoprecipitation, Concentration Assay, Two Tailed Test

Journal: Life Science Alliance

Article Title: α-Actinin-4 recruits Shp2 into focal adhesions to potentiate ROCK2 activation in podocytes

doi: 10.26508/lsa.202201557

Figure Lengend Snippet: MEFs seeded on FN-coated dishes were treated with or without FAK inhibitor 14 (15 μM) for 90 min for isolation of FAs as . The isolated FA fractions (FA) were subjected to Western analysis as indicated. Relative FAK Y397 phosphorylation and levels of Shp2 versus vinculin in FA fractions were measured and showed in .

Article Snippet: Anti-paxillin antibody and mouse collagen type-IV from BD Biosciences; anti-β-actin, anti-β-tubulin, and anti-flag antibodies, FITC-phalloidin, ROCK inhibitor Y27632, and Shp2 inhibitor IIB-08 from Sigma-Aldrich; FAK inhibitor 14 from Tocris Bioscience, anti-pY397FAK, anti-pY31-paxillin, and anti-ZO-1 antibodies from Invitrogen; anti-pY542 Shp2, anti-pT18/pS19-MLC, and anti-MLC antibodies from Cell Signaling; anti-α-actinin-1 antibody from Chemicon; anti-α-actinin-4 and anti-phosphotyrosine (clone 4G10) antibodies from Millipore; anti-FAK, anti-Shp2, anti-ROCK1, and anti-ROCK2, antibodies from Santa Cruz Biotechnology Inc.; anti-synaptopodin antibody from Novus; anti-podocin antibody from Abcam; λ protein phosphatase from New England BioLabs.

Techniques: Isolation, Western Blot, Phospho-proteomics

Journal: Life Science Alliance

Article Title: α-Actinin-4 recruits Shp2 into focal adhesions to potentiate ROCK2 activation in podocytes

doi: 10.26508/lsa.202201557

Figure Lengend Snippet: (A, B) HEK293T cells seeded were co-transfected with the expression constructs of flag-Shp2 and GFP-α-actinin-4 (A) or mCherry-α-actinin-1 (B). Cells were harvested for immunoprecipitation with anti-flag, and the immunoprecipitated proteins were analyzed by Western blot as indicated. (C) HEK293T cells were transiently transfected with the expression construct of GFP-α-actinin-4. Cells were harvested for immunoprecipitation with the anti-GFP antibody (GFP-trap) and then subjected to Western blot analysis with anti-pY (4G10) and anti-GFP antibodies.

Article Snippet: Anti-paxillin antibody and mouse collagen type-IV from BD Biosciences; anti-β-actin, anti-β-tubulin, and anti-flag antibodies, FITC-phalloidin, ROCK inhibitor Y27632, and Shp2 inhibitor IIB-08 from Sigma-Aldrich; FAK inhibitor 14 from Tocris Bioscience, anti-pY397FAK, anti-pY31-paxillin, and anti-ZO-1 antibodies from Invitrogen; anti-pY542 Shp2, anti-pT18/pS19-MLC, and anti-MLC antibodies from Cell Signaling; anti-α-actinin-1 antibody from Chemicon; anti-α-actinin-4 and anti-phosphotyrosine (clone 4G10) antibodies from Millipore; anti-FAK, anti-Shp2, anti-ROCK1, and anti-ROCK2, antibodies from Santa Cruz Biotechnology Inc.; anti-synaptopodin antibody from Novus; anti-podocin antibody from Abcam; λ protein phosphatase from New England BioLabs.

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

Journal: Life Science Alliance

Article Title: α-Actinin-4 recruits Shp2 into focal adhesions to potentiate ROCK2 activation in podocytes

doi: 10.26508/lsa.202201557

Figure Lengend Snippet: (A) Wild-type (WT) and Ptpn11 Ex3−/− MEFs were seeded on FN-coated coverslips for 2 h and fixed for in situ proximity ligation assay (PLA) with anti-Shp2 plus anti-α-actinin-4 antibodies (red). Reaction with anti-Shp2 antibody serves as a negative control. After reaction, cells were stained with FITC-phalloidin (green) and Hoechst (blue) for F-actin and nucleus, respectively. Scatter dot plots show the percentage of cells with PLA count >5 in each independent experiments. (B) MEFs seeded FN-coated coverslips were treated with or without FAK inhibitor 14 (20 μM) for PLA assay. Scatter dot plots (mean ± SD) show PLA counts from more than 36 cells in three independent experiments; each dot represents one single cell. **** P < 0.0001 (Mann–Whitney U test). Scale bars, 10 μm.

Article Snippet: Anti-paxillin antibody and mouse collagen type-IV from BD Biosciences; anti-β-actin, anti-β-tubulin, and anti-flag antibodies, FITC-phalloidin, ROCK inhibitor Y27632, and Shp2 inhibitor IIB-08 from Sigma-Aldrich; FAK inhibitor 14 from Tocris Bioscience, anti-pY397FAK, anti-pY31-paxillin, and anti-ZO-1 antibodies from Invitrogen; anti-pY542 Shp2, anti-pT18/pS19-MLC, and anti-MLC antibodies from Cell Signaling; anti-α-actinin-1 antibody from Chemicon; anti-α-actinin-4 and anti-phosphotyrosine (clone 4G10) antibodies from Millipore; anti-FAK, anti-Shp2, anti-ROCK1, and anti-ROCK2, antibodies from Santa Cruz Biotechnology Inc.; anti-synaptopodin antibody from Novus; anti-podocin antibody from Abcam; λ protein phosphatase from New England BioLabs.

Techniques: In Situ, Proximity Ligation Assay, Negative Control, Staining, MANN-WHITNEY

Journal: Life Science Alliance

Article Title: α-Actinin-4 recruits Shp2 into focal adhesions to potentiate ROCK2 activation in podocytes

doi: 10.26508/lsa.202201557

Figure Lengend Snippet: (A) The modified DNA sequence of Actn4 in two selected Actn4 −/− MEF clones. The gRNA targeted region is highlighted in yellow; PAN site is shown in light blue, and modified DNA sequence is shown in red. The expression of α-actinin-4 in these clones was checked by Western blotting. (B) The FRET efficiency images of a wild-type or Actn4 −/− MEF transfected with Shp2 FRET reporter (Shp2-SWAP). (C) The expression construct of mApple-Actn4 (red) was co-transfected with Shp2-SWAP into MEF cells for FRET imaging analysis. (D) Scatter dot plots (mean ± SD) of the Shp2-SWAP FRET efficiency. The sample numbers are 24 and 17 for WT 16 and 18 for Actn4 −/− #1 and 25 and 15 and 24 for Actn4 −/− #2 cells in the absent or present of mApple-Actn4 co-transfection, respectively. Differences between continuous variables were compared using the Mann–Whitney U test. *** P < 0.0005. Scale bars, 10 μm. Source data are available for this figure.

Article Snippet: Anti-paxillin antibody and mouse collagen type-IV from BD Biosciences; anti-β-actin, anti-β-tubulin, and anti-flag antibodies, FITC-phalloidin, ROCK inhibitor Y27632, and Shp2 inhibitor IIB-08 from Sigma-Aldrich; FAK inhibitor 14 from Tocris Bioscience, anti-pY397FAK, anti-pY31-paxillin, and anti-ZO-1 antibodies from Invitrogen; anti-pY542 Shp2, anti-pT18/pS19-MLC, and anti-MLC antibodies from Cell Signaling; anti-α-actinin-1 antibody from Chemicon; anti-α-actinin-4 and anti-phosphotyrosine (clone 4G10) antibodies from Millipore; anti-FAK, anti-Shp2, anti-ROCK1, and anti-ROCK2, antibodies from Santa Cruz Biotechnology Inc.; anti-synaptopodin antibody from Novus; anti-podocin antibody from Abcam; λ protein phosphatase from New England BioLabs.

Techniques: Modification, Sequencing, Clone Assay, Expressing, Western Blot, Transfection, Construct, Imaging, Cotransfection, MANN-WHITNEY

Journal: Life Science Alliance

Article Title: α-Actinin-4 recruits Shp2 into focal adhesions to potentiate ROCK2 activation in podocytes

doi: 10.26508/lsa.202201557

Figure Lengend Snippet: MEFs were transfected with Shp2 FRET reporter (Shp2-SWAP) and then replated on fibronectin (FN) or poly–L-lysine (PLL)–coated glass bottom dishes for FRET imaging. Scatter dot plots show the Shp2-SWAP FRET efficiency of each individual cells. Data are expressed as mean ± SD from more than 10 cells in two independent experiments. Differences between continuous variables were compared using the Mann–Whitney U test. ** P < 0.005. Scale bar, 10 μm.

Article Snippet: Anti-paxillin antibody and mouse collagen type-IV from BD Biosciences; anti-β-actin, anti-β-tubulin, and anti-flag antibodies, FITC-phalloidin, ROCK inhibitor Y27632, and Shp2 inhibitor IIB-08 from Sigma-Aldrich; FAK inhibitor 14 from Tocris Bioscience, anti-pY397FAK, anti-pY31-paxillin, and anti-ZO-1 antibodies from Invitrogen; anti-pY542 Shp2, anti-pT18/pS19-MLC, and anti-MLC antibodies from Cell Signaling; anti-α-actinin-1 antibody from Chemicon; anti-α-actinin-4 and anti-phosphotyrosine (clone 4G10) antibodies from Millipore; anti-FAK, anti-Shp2, anti-ROCK1, and anti-ROCK2, antibodies from Santa Cruz Biotechnology Inc.; anti-synaptopodin antibody from Novus; anti-podocin antibody from Abcam; λ protein phosphatase from New England BioLabs.

Techniques: Transfection, Imaging, MANN-WHITNEY

Journal: Life Science Alliance

Article Title: α-Actinin-4 recruits Shp2 into focal adhesions to potentiate ROCK2 activation in podocytes

doi: 10.26508/lsa.202201557

Figure Lengend Snippet: (A) The modified DNA sequence of two selected Actn4 −/− podocyte clones are showed. The gRNA targeted region is highlighted in yellow; the PAN site is shown in light blue, and modified DNA sequence is shown in red. Podocytes maintained at permission (33°C) or differentiation (37°C) conditions for 14 d were harvested for Western blotting analysis as indicated. (B) The phosphorylation status of Shp2 at Y542 in these clones were also detected. (C) Podocytes were induced for differentiation and then maintained in attached (Att) or suspension for 30 min (Sus) before harvested for Western blotting analysis as indicated. (D) Podocytes were induced for differentiation and seeded on collagen type-IV–coated glass coverslips for immunofluorescence staining with anti-paxillin antibody and Hoechst for detecting FA and DNA, respectively. Scatter dot plots of the numbers of small FA (area < 1 μm 2 ) and matured FA (area > 1 μm 2 ) were shown. Data are expressed as mean ± SD from 30 representative cells in three independent experiments. Differences between continuous variables were compared using two-tail unpaired student t test. *** P < 0.001. ns, not statistically significant. Bars, 10 μm. Source data are available for this figure.

Article Snippet: Anti-paxillin antibody and mouse collagen type-IV from BD Biosciences; anti-β-actin, anti-β-tubulin, and anti-flag antibodies, FITC-phalloidin, ROCK inhibitor Y27632, and Shp2 inhibitor IIB-08 from Sigma-Aldrich; FAK inhibitor 14 from Tocris Bioscience, anti-pY397FAK, anti-pY31-paxillin, and anti-ZO-1 antibodies from Invitrogen; anti-pY542 Shp2, anti-pT18/pS19-MLC, and anti-MLC antibodies from Cell Signaling; anti-α-actinin-1 antibody from Chemicon; anti-α-actinin-4 and anti-phosphotyrosine (clone 4G10) antibodies from Millipore; anti-FAK, anti-Shp2, anti-ROCK1, and anti-ROCK2, antibodies from Santa Cruz Biotechnology Inc.; anti-synaptopodin antibody from Novus; anti-podocin antibody from Abcam; λ protein phosphatase from New England BioLabs.

Techniques: Modification, Sequencing, Clone Assay, Western Blot, Phospho-proteomics, Suspension, Immunofluorescence, Staining

Journal: Life Science Alliance

Article Title: α-Actinin-4 recruits Shp2 into focal adhesions to potentiate ROCK2 activation in podocytes

doi: 10.26508/lsa.202201557

Figure Lengend Snippet: (A) Wild-type and Actn4 −/− podocytes maintained at permission (33°C) or differentiation (37°C) conditions on type-IV collagen–coated glass coverslips were fixed for in situ proximity ligation assay with anti-Shp2 plus anti-α-actinin-4 antibodies (red). After reaction, cells were stained with FITC-phalloidin (green) and Hoechst (blue) for F-actin and nucleus, respectively. (B) Differentiated podocytes were fixed for immunofluorescence staining with anti-ZO-1 antibody and Hoechst for detecting intercellular junctions and DNA, respectively. Scale bar, 10 μm.

Article Snippet: Anti-paxillin antibody and mouse collagen type-IV from BD Biosciences; anti-β-actin, anti-β-tubulin, and anti-flag antibodies, FITC-phalloidin, ROCK inhibitor Y27632, and Shp2 inhibitor IIB-08 from Sigma-Aldrich; FAK inhibitor 14 from Tocris Bioscience, anti-pY397FAK, anti-pY31-paxillin, and anti-ZO-1 antibodies from Invitrogen; anti-pY542 Shp2, anti-pT18/pS19-MLC, and anti-MLC antibodies from Cell Signaling; anti-α-actinin-1 antibody from Chemicon; anti-α-actinin-4 and anti-phosphotyrosine (clone 4G10) antibodies from Millipore; anti-FAK, anti-Shp2, anti-ROCK1, and anti-ROCK2, antibodies from Santa Cruz Biotechnology Inc.; anti-synaptopodin antibody from Novus; anti-podocin antibody from Abcam; λ protein phosphatase from New England BioLabs.

Techniques: In Situ, Proximity Ligation Assay, Staining, Immunofluorescence

Journal: Oncotarget

Article Title: Shp2 SUMOylation promotes ERK activation and hepatocellular carcinoma development

doi:

Figure Lengend Snippet: (A) 293T cells were transfected with HA-Shp2, Flag-Ubc9 along with His-SUMO1, RH-SUMO2 or RH-SUMO3. SUMOylated proteins were purified by using Ni 2+ -NTA affinity pull down and SUMOylated Shp2 was immunoblotted with anti-HA antibody. (B) 293T cells were transfected with HA-Shp2, along with or without His-SUMO1 and Flag-Ubc9, and SUMOylated Shp2 was detected by the method of in vivo SUMOylation assay using Ni 2+ -NTA agarose beads. (C) Lysates from 293T cells expressing Flag-Shp2, His-SUMO1, Flag-Ubc9 with or without SENP1 were pulled down treated with Ni 2+ -NTA agarose beads for SUMOylation assays. (D) Stable SENP1-knockdown 293T cells were used to confirm Shp2 SUMOylation by immunoblotting with anti-HA of Ni 2+ -NTA precipitates. (E) The pGEX4T1-Shp2 (human Shp2, left panel; mouse Shp2, right panel) plasmids were co-transformed with or without pE1E2SUMO1 into E.coli BL21 (DE3), proteins were purified with GST agarose beads followed by Western blotting analysis for SUMOylation in vitro . (F) Lysates from SENP +/+ or SENP1 −/− mouse brain tissues at embryonic day 13.5 were immunoprecipitated with anti-SUMO1 antibody, and then immunoblotted with anti-Shp2 and anti-SUMO1 antibodies. Lysates as Input were Western blotted with anti-Shp2, anti-Senp1 and anti-β-Actin antibodies.

Article Snippet: Antibodies against Shp2 (B-1), IGF-IRβ (H-60), Gab1 (H198) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA); antibodies against phospho-Shp2 pY542 (#2184–1), phospho-EGFR pY1068 (#1138–1) were from Epitomics (Burlingame, CA, USA).

Techniques: Transfection, Purification, In Vivo, Expressing, Knockdown, Western Blot, Transformation Assay, In Vitro, Immunoprecipitation

Journal: Oncotarget

Article Title: Shp2 SUMOylation promotes ERK activation and hepatocellular carcinoma development

doi:

Figure Lengend Snippet: (A) Schematic maps of Shp2 domains. N-SH2 (1–106 aa), N-terminal Src Homology 2 domain; C-SH2 (107–220 aa), C-terminal Src Homology 2 domain; ΔSH2: deletion of amino acids 1–209 containing N-SH2 and C-SH2 domain regions. (B–C) pEF-HA-N-SH2, -C-SH2 (B), or −ΔSH2 (C) were co-transfected with Flag-Ubc9 and His-SUMO1 into 293T cells, and then SUMOylation assays by using Ni 2+ -NTA agarose beads were performed as above. (D) 293T cells co-transfected with ΔSH2 WT or mutant ΔSH2 K590R with or without Flag-Ubc9 and His-SUMO1 were lysed and treated for SUMOylation assays. (E) 293T cells co-transfected with full-longth Shp2 WT or Shp2 K590R with or without Flag-Ubc9 and His-SUMO1 were lysed and treated for SUMOylation assays. (F) The GST-Shp2 WT or GST-Shp2 K590R plasmid was co-transformed with pE1E2SUMO1 into E.coli BL21 (DE3). Immunoblotting was conducted after GST pull down.

Article Snippet: Antibodies against Shp2 (B-1), IGF-IRβ (H-60), Gab1 (H198) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA); antibodies against phospho-Shp2 pY542 (#2184–1), phospho-EGFR pY1068 (#1138–1) were from Epitomics (Burlingame, CA, USA).

Techniques: Transfection, Mutagenesis, Plasmid Preparation, Transformation Assay, Western Blot

Journal: Oncotarget

Article Title: Shp2 SUMOylation promotes ERK activation and hepatocellular carcinoma development

doi:

Figure Lengend Snippet: (A) 293T cells stably expressing Shp2 WT or Shp2 K590R were starved overnight and then stimulated by EGF for 5 and 10 min. Cell lysates were used for immunoblotting analysis of phospho-ERK1/2, ERK1/2, HA-Shp2 and GAPDH. (B–C) Endogenous Shp2 in HepG2 and SMMC-7721 were knockdown by a short hairpin RNA targeting 3′-UTR of ptpn11 mRNA (shShp2) by using lentiviral pGreenPuro system. Shp2 WT and Shp2 K590R were reintroduced respectively into stable HepG2-shShp2 (B) and SMMC-7721-shShp2 (C) cells. After serum-starvation overnight, stable HepG2 and SMMC-7721 cell lines were stimulated with 100 ng/mL of EGF for 5 min, and then the ERK activities were determined by Western blotting (left panels). The data are presented as the mean ± s.d. ( n = 3) (right panels). (D) Soft agar colony-forming assays, stable HepG2-shShp2 and re-expressing Shp2 WT or Shp2 K590R cell lines were seeded in 2 mL of medium containing 5% FBS with 0.35% agar at 5 × 10 3 cells/well and layered onto the base. The photographs were taken 20 days later and the number of colonies was scored. Each value represents the mean ± s.e.m. of three independent experiments with triplicates each. (E) Stable SMMC-7721-shShp2 re-expressing Shp2 WT or Shp2 K590R cell lines were injected subcutaneously into male BALB/c nude mice ( n = 5) individually. The sizes of tumors were measured at day 9, 12 and 15 days after injection (left panel) and the tumors were weighed (right panel).

Article Snippet: Antibodies against Shp2 (B-1), IGF-IRβ (H-60), Gab1 (H198) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA); antibodies against phospho-Shp2 pY542 (#2184–1), phospho-EGFR pY1068 (#1138–1) were from Epitomics (Burlingame, CA, USA).

Techniques: Stable Transfection, Expressing, Western Blot, Knockdown, shRNA, Injection

Journal: Oncotarget

Article Title: Shp2 SUMOylation promotes ERK activation and hepatocellular carcinoma development

doi:

Figure Lengend Snippet: (A) SMMC-7721-shShp2 cells stably re-expressing Shp2 WT , Shp2 K590R or -truncated form (amino acids 1–587) were starved overnight, and then stimulated by EGF for 5 min. Cell lysates were used for immunoblotting analysis of phospho-ERK1/2, ERK1/2 and GAPDH (left panels). The data are presented as the mean ± s.d. ( n = 3) (right panels). (B) HepG2-shShp2 cells stably re-expressing Shp2 WT , Shp2 K590R or Shp2 K590R -SUMO1 (a fusion construct) were stimulated with EGF for 5 min as before, and then the ERK activities were determined by Western blotting (left panels). The data are presented as the mean ± s.d. ( n = 3) (right panels). (C) Endogenous SUMO1 in SMMC 7721 and HepG2 was knockdown by a short hairpin RNA targeting 3′-UTR of SUMO1 mRNA (shSUMO1) by using lentiviral vector pLKO.1 system, and the ERK1/2 activities were determined. (D) SMMC-7721-shSUMO1 and HepG2-shSUMO1 stably expressing Shp2 WT or Shp2 K590R cells were serum-starved, and then stimulated with 100 ng/mL of EGF for 5 min, and the ERK activities were determined by Western blotting. (E) Soft agar colony forming assays, HepG2-shSUMO1 and SMMC-7721-shSUMO1 stably expressing Shp2 WT or Shp2 K590R cells were seeded in 2 ml of medium containing 5% FBS with 0.35% agar at 1 × 10 4 and 2 × 10 3 cells/well, respectively. The photographs were taken 20 days later and the number of colonies was scored. Each value represents the mean ± s.e.m. of three independent experiments with triplicates each.

Article Snippet: Antibodies against Shp2 (B-1), IGF-IRβ (H-60), Gab1 (H198) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA); antibodies against phospho-Shp2 pY542 (#2184–1), phospho-EGFR pY1068 (#1138–1) were from Epitomics (Burlingame, CA, USA).

Techniques: Stable Transfection, Expressing, Western Blot, Construct, Knockdown, shRNA, Plasmid Preparation

Journal: Oncotarget

Article Title: Shp2 SUMOylation promotes ERK activation and hepatocellular carcinoma development

doi:

Figure Lengend Snippet: (A) In vivo Ni 2+ -NTA resin pull-down SUMOylation assays for human and mouse Shp2 in 293T cells. (B) In vitro GST agarose pull-down SUMOylation assays for human and mouse Shp2 with pE1E2SUMO1 into E.coli BL21 (DE3). (C) SUMOylation assays for mouse Shp2-WT and -R594K mutant in 293T cells by using the method of Ni 2+ -NTA resin pull-down. (D) Immunoblotting analysis of ERK1/2 phosphorylation of stable 293T cell lines expressing hShp2 WT , hShp2 K590R , mShp2 WT and mShp2 R594K . (E–F) Serum-starved 293T cells transiently expressing hShp2 or mShp2 were stimulated with 100 ng/mL of EGF for 5 min and the ERK activities were determined by Western blotting (E); the same lysates were used for immunoprecipitation with anti-HA, then performed the Shp2 phosphatase activity assays (F). (G–H) Serum-starved 293T cells transiently expressing mShp2 WT or mShp2 R594K were stimulated with 100 ng/mL of EGF for 5 or 10 min and the ERK activities were determined by Western blotting (G); the same lysates (5 min) were used for immunoprecipitation with anti-HA, then performed the Shp2 phosphatase activity assays (H).

Article Snippet: Antibodies against Shp2 (B-1), IGF-IRβ (H-60), Gab1 (H198) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA); antibodies against phospho-Shp2 pY542 (#2184–1), phospho-EGFR pY1068 (#1138–1) were from Epitomics (Burlingame, CA, USA).

Techniques: In Vivo, In Vitro, Mutagenesis, Western Blot, Phospho-proteomics, Expressing, Immunoprecipitation, Activity Assay

Journal: Oncotarget

Article Title: Shp2 SUMOylation promotes ERK activation and hepatocellular carcinoma development

doi:

Figure Lengend Snippet: (A) Cytosolic fractions and membranous fractions extracted from EGF-stimulated SMMC-7721-shShp2 cells stably re-expressing hShp2 WT or hShp2 K590R were analyzed by Western blotting with antibodies against HA, IGF-1Rβ (as a membrane protein marker) and GAPDH (as a cytosolic marker). (B) 293T or 293T-shSENP1 cells were co-transfected with HA-Shp2 and Flag-Gab1 plasmids. 24 h after transfection, cells were subjected to serum deprivation for 16 h, followed by the treatment with 100 ng/mL of EGF for 5 min. Cell lysates were immunoprecipitated and subsequently immunoblotted with indicated antibodies. (C) Lysates from EGF-treated SMMC-7721-shShp2 cells stably re-expressing hShp2 WT or hShp2 K590R were immunoprecipitated with anti-Gab1 antibody and then immunoblotted with anti-HA antibody. Lysates were also used as Input for immunoblotting with antibodies against pERK1/2 and other indicated. (D) Lysates from EGF-treated SMMC-7721-shSUMO1 cells stably re-expressing hShp2 WT or hShp2 K590R were immunoprecipitated with anti-Gab1 antibody and then immunoblotted with anti-HA antibody. Lysates were also used as Input for immunoblotting with antibodies indicated. (E) Flag-Gab1 was transiently expressed in 293T cells and GST-Shp2 with pE1E2SUMO1 were transformed into E.coli BL21 (DE3). Two reciprocal pull-down assays of GST-proteins (left panels) and anti-Flag/IP (right panels) were performed, and then immunoblotted. (F) Lysates from 293T cells co-transfected with His-SUMO1 and Flag-Gab1 WT or Flag-Gab1 SIM1/2mut palsmids were immunoprecipitated with anti-Flag antibody, and then immunoblotted with anti-His antibody. Lysates were also used as Input for immunoblotting with indicated antibodies (left panels). The consensus amino acid sequences (yellow labeled) of predicted SIM1 and SIM2 of Gab1 were mutated to alanine (right panels).

Article Snippet: Antibodies against Shp2 (B-1), IGF-IRβ (H-60), Gab1 (H198) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA); antibodies against phospho-Shp2 pY542 (#2184–1), phospho-EGFR pY1068 (#1138–1) were from Epitomics (Burlingame, CA, USA).

Techniques: Stable Transfection, Expressing, Western Blot, Membrane, Marker, Transfection, Immunoprecipitation, Transformation Assay, Labeling

Journal: Oncotarget

Article Title: Shp2 SUMOylation promotes ERK activation and hepatocellular carcinoma development

doi:

Figure Lengend Snippet: Shp2 SUMOyaltion promotes activation of the ERK signaling via facilitating the formation of Shp2-Gab1 complex and thereby accelerates HCC cell growth.

Article Snippet: Antibodies against Shp2 (B-1), IGF-IRβ (H-60), Gab1 (H198) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA); antibodies against phospho-Shp2 pY542 (#2184–1), phospho-EGFR pY1068 (#1138–1) were from Epitomics (Burlingame, CA, USA).

Techniques: Activation Assay