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3CL pro cleaves RPAP1 and PTBP1, altering PTBP1 localization (A) Locations of 3CL pro cleavage sites in RPAP1 and PTBP1 identified by TAILS neo-N-terminal peptides (red) and Edman sequencing (green). Representative MS/MS spectra of cleaved neo-N-terminal peptides. (B) SDS-PAGE and Edman sequencing of human recombinant RPAP1 and PTBP1 incubated with 3CL pro +/− inhibitor GC376, or 3CL pro -C145A (1:5 mol/mol, E:S). Δ-substrate, no sequence obtained. (C) MALDI-TOF-MS kinetic analyses of 3CL pro cleavage of synthetic P4–P4’ peptides. (D) RPAP1 and PTBP1 immunoblots of primary HAECs lysates from 5 donors incubated with 3CL pro or 3CL pro -C145A (1:200 w/w, E:S) for 18 h, 37°C. (E and F) (E) Immunoblots of 3CL pro , N-protein and (F) PTBP1 of Vero E6 cells infected with SARS-CoV-2 at a MOI 0.1 for 24 (n = 4) and 48 (n = 4) hpi, or mock (n = 3) ∗ , unspecific bands. (G) Subcellular localization of PTBP1 by confocal imaging. SARS-CoV-2-infected Vero E6 cells (N = 5, scale bar 50 μm). Cyan boxes, Spike-negative (S–) uninfected cells. Green boxes, Spike-positive (S+) infected cells. Enlarged detail of mock-infected, S+ and S– cells in the same field, scale bar 10 μm. (H) Nuclear to cytoplasmic ratio of PTBP1 was quantified for mock and Spike-positive SARS-CoV-2 infected cells. Statistical significance was assessed by Student’s t test (mean ± SD, N = 5, n = 51 cells, ∗ p ≤ 0.05). β-actin <t>and</t> <t>β-tubulin</t> loading controls. See also .
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microscope axio imager.d2  (Carl Zeiss)
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3CL pro cleaves RPAP1 and PTBP1, altering PTBP1 localization (A) Locations of 3CL pro cleavage sites in RPAP1 and PTBP1 identified by TAILS neo-N-terminal peptides (red) and Edman sequencing (green). Representative MS/MS spectra of cleaved neo-N-terminal peptides. (B) SDS-PAGE and Edman sequencing of human recombinant RPAP1 and PTBP1 incubated with 3CL pro +/− inhibitor GC376, or 3CL pro -C145A (1:5 mol/mol, E:S). Δ-substrate, no sequence obtained. (C) MALDI-TOF-MS kinetic analyses of 3CL pro cleavage of synthetic P4–P4’ peptides. (D) RPAP1 and PTBP1 immunoblots of primary HAECs lysates from 5 donors incubated with 3CL pro or 3CL pro -C145A (1:200 w/w, E:S) for 18 h, 37°C. (E and F) (E) Immunoblots of 3CL pro , N-protein and (F) PTBP1 of Vero E6 cells infected with SARS-CoV-2 at a MOI 0.1 for 24 (n = 4) and 48 (n = 4) hpi, or mock (n = 3) ∗ , unspecific bands. (G) Subcellular localization of PTBP1 by confocal imaging. SARS-CoV-2-infected Vero E6 cells (N = 5, scale bar 50 μm). Cyan boxes, Spike-negative (S–) uninfected cells. Green boxes, Spike-positive (S+) infected cells. Enlarged detail of mock-infected, S+ and S– cells in the same field, scale bar 10 μm. (H) Nuclear to cytoplasmic ratio of PTBP1 was quantified for mock and Spike-positive SARS-CoV-2 infected cells. Statistical significance was assessed by Student’s t test (mean ± SD, N = 5, n = 51 cells, ∗ p ≤ 0.05). β-actin and β-tubulin loading controls. See also .

Journal: Cell Reports

Article Title: Mechanistic insights into COVID-19 by global analysis of the SARS-CoV-2 3CL pro substrate degradome

doi: 10.1016/j.celrep.2021.109892

Figure Lengend Snippet: 3CL pro cleaves RPAP1 and PTBP1, altering PTBP1 localization (A) Locations of 3CL pro cleavage sites in RPAP1 and PTBP1 identified by TAILS neo-N-terminal peptides (red) and Edman sequencing (green). Representative MS/MS spectra of cleaved neo-N-terminal peptides. (B) SDS-PAGE and Edman sequencing of human recombinant RPAP1 and PTBP1 incubated with 3CL pro +/− inhibitor GC376, or 3CL pro -C145A (1:5 mol/mol, E:S). Δ-substrate, no sequence obtained. (C) MALDI-TOF-MS kinetic analyses of 3CL pro cleavage of synthetic P4–P4’ peptides. (D) RPAP1 and PTBP1 immunoblots of primary HAECs lysates from 5 donors incubated with 3CL pro or 3CL pro -C145A (1:200 w/w, E:S) for 18 h, 37°C. (E and F) (E) Immunoblots of 3CL pro , N-protein and (F) PTBP1 of Vero E6 cells infected with SARS-CoV-2 at a MOI 0.1 for 24 (n = 4) and 48 (n = 4) hpi, or mock (n = 3) ∗ , unspecific bands. (G) Subcellular localization of PTBP1 by confocal imaging. SARS-CoV-2-infected Vero E6 cells (N = 5, scale bar 50 μm). Cyan boxes, Spike-negative (S–) uninfected cells. Green boxes, Spike-positive (S+) infected cells. Enlarged detail of mock-infected, S+ and S– cells in the same field, scale bar 10 μm. (H) Nuclear to cytoplasmic ratio of PTBP1 was quantified for mock and Spike-positive SARS-CoV-2 infected cells. Statistical significance was assessed by Student’s t test (mean ± SD, N = 5, n = 51 cells, ∗ p ≤ 0.05). β-actin and β-tubulin loading controls. See also .

Article Snippet: The primary antibodies and dilutions used were: mouse monoclonal anti-SARS-CoV-2 nucleocapsid antibody (1:1,000, Invitrogen, MA5-29981, RRID: AB_2785780 ); rabbit anti-SARS-CoV-1 3CL pro antibody (1:2000, Rockland, 200-401-A51, RRID: AB_828457 ); rabbit polyclonal anti-RPAP1 antibody (1:1,000, Proteintech, 15138-1-AP, RRID: AB_2301137 ); mouse monoclonal anti-PTBP1 antibody (1:500, Biolegend, 630101, 3H7, RRID: AB_2171285 ); rabbit polyclonal anti-MAP4K5 antibody (1:1,000, Cusabio, CSB-PA013440DSR2HU, RRID: AB_2892084 ); rabbit polyclonal anti-CREB1 antibody (1:1,000, Abclonal, A11989, RRID: AB_2758916 ); rabbit polyclonal anti-YAP1 antibody (1:1,000, Abclonal, A11430, RRID: AB_2758556 ); rabbit polyclonal anti-FYCO1 antibody (1:1,000, Cusabio, CSB-PA866262LA01HU, RRID: AB_2892085 ); rabbit polyclonal anti-FAF1 antibody (1:1,000, Abclonal, A2921, RRID: AB_2764739 ); goat polyclonal anti-Gal8 antibody (1:400, R&D Systems, AF1305, RRID: AB_2137229 ); rabbit polyclonal anti-KPNA3 (IMA4) antibody (1:1,000, Abclonal, A8347, RRID: AB_2770124 ); rabbit polyclonal anti-NUP107 antibody (1:1,000, Abclonal, A13110, RRID: AB_2759959 ); mouse monoclonal anti-IRS2 antibody (1:300, R&D Systems, MAB6347, 676415, RRID: AB_10992928 ); mouse monoclonal anti-FLAG M2 antibody (1:10,000, Sigma, F3165, RRID: AB_259529 ); mouse monoclonal anti-β-tubulin antibody (1:2000, AbLab, 21-0018-00, clone BT7R); mouse monoclonal anti-β-actin antibody (1:1,000, Abcam, ab8226, RRID: AB_306371 ); rabbit monoclonal anti-β-actin antibody (1:200, Abcam, ab115777, RRID: AB_10899528 ).

Techniques: Sequencing, Tandem Mass Spectroscopy, SDS Page, Recombinant, Incubation, Western Blot, Infection, Imaging

Hippo pathway substrate validation (A) 3CL pro cleavage sites in MAP4K5 and CREB1 identified by TAILS neo-N-terminal peptides (red) and Edman sequencing (green). Representative MS/MS spectra of cleaved neo-N-terminal peptides. (B) MALDI-TOF-MS kinetic analyses of 3CL pro cleavage of P4–P4’ peptides of MAP4K5 and CREB1. (C) SDS-PAGE, Edman sequencing (green) and immunoblot validation of human MAP4K5 and CREB1 substrates incubated with 3CL pro +/− inhibitor GC376, or 3CL pro -C145A (1:5 mol/mol, E:S). ΔMAP4K5 or ΔCREB1, no sequence obtained. (D and H) (D) YAP1, MAP4K5, CREB1 and (H) FYCO1 and FAF1 immunoblots of lysates from primary HAECs incubated with 3CL pro or 3CL pro -C145A (1:200 w/w, E:S) for 18 h, 37°C. (E and F) (E) YAP1 and (F) MAP4K5 immunoblots of Vero E6 cells at 24 (n = 4) and 48 (n = 4) hpi (MOI 0.1) or mock (n = 3). MAP4K5 activity assay measured as ATP consumption using myelin basic protein as substrate. The area under the curve was calculated and compared by Student’s t test, (mean ± SD, n = 2, ∗∗ p ≤ 0.01). (G, I, and J) (G) Lysates of human Calu-3 lung cells infected with SARS-CoV-2 (MOI 0.1 and 1.0, n = 4, mock n = 2) were immunoblotted for (G) CREB1 48 hpi, (I) FYCO1 24 hpi and (J) FAF1 48 hpi. Statistical analysis of the relative amount of full-length protein (E and F) or proteolytic bands (G, I, and J) identified by molecular weights relative to β-actin was assessed by one-way ANOVA and Dunnett’s multiple comparisons test. Box and whiskers (min to max) plots, ∗∗∗ p ≤ 0.001, ∗∗ p ≤ 0.01, ∗ p ≤ 0.05, ns p > 0.05. β-actin and β-tubulin loading controls. See also and .

Journal: Cell Reports

Article Title: Mechanistic insights into COVID-19 by global analysis of the SARS-CoV-2 3CL pro substrate degradome

doi: 10.1016/j.celrep.2021.109892

Figure Lengend Snippet: Hippo pathway substrate validation (A) 3CL pro cleavage sites in MAP4K5 and CREB1 identified by TAILS neo-N-terminal peptides (red) and Edman sequencing (green). Representative MS/MS spectra of cleaved neo-N-terminal peptides. (B) MALDI-TOF-MS kinetic analyses of 3CL pro cleavage of P4–P4’ peptides of MAP4K5 and CREB1. (C) SDS-PAGE, Edman sequencing (green) and immunoblot validation of human MAP4K5 and CREB1 substrates incubated with 3CL pro +/− inhibitor GC376, or 3CL pro -C145A (1:5 mol/mol, E:S). ΔMAP4K5 or ΔCREB1, no sequence obtained. (D and H) (D) YAP1, MAP4K5, CREB1 and (H) FYCO1 and FAF1 immunoblots of lysates from primary HAECs incubated with 3CL pro or 3CL pro -C145A (1:200 w/w, E:S) for 18 h, 37°C. (E and F) (E) YAP1 and (F) MAP4K5 immunoblots of Vero E6 cells at 24 (n = 4) and 48 (n = 4) hpi (MOI 0.1) or mock (n = 3). MAP4K5 activity assay measured as ATP consumption using myelin basic protein as substrate. The area under the curve was calculated and compared by Student’s t test, (mean ± SD, n = 2, ∗∗ p ≤ 0.01). (G, I, and J) (G) Lysates of human Calu-3 lung cells infected with SARS-CoV-2 (MOI 0.1 and 1.0, n = 4, mock n = 2) were immunoblotted for (G) CREB1 48 hpi, (I) FYCO1 24 hpi and (J) FAF1 48 hpi. Statistical analysis of the relative amount of full-length protein (E and F) or proteolytic bands (G, I, and J) identified by molecular weights relative to β-actin was assessed by one-way ANOVA and Dunnett’s multiple comparisons test. Box and whiskers (min to max) plots, ∗∗∗ p ≤ 0.001, ∗∗ p ≤ 0.01, ∗ p ≤ 0.05, ns p > 0.05. β-actin and β-tubulin loading controls. See also and .

Article Snippet: The primary antibodies and dilutions used were: mouse monoclonal anti-SARS-CoV-2 nucleocapsid antibody (1:1,000, Invitrogen, MA5-29981, RRID: AB_2785780 ); rabbit anti-SARS-CoV-1 3CL pro antibody (1:2000, Rockland, 200-401-A51, RRID: AB_828457 ); rabbit polyclonal anti-RPAP1 antibody (1:1,000, Proteintech, 15138-1-AP, RRID: AB_2301137 ); mouse monoclonal anti-PTBP1 antibody (1:500, Biolegend, 630101, 3H7, RRID: AB_2171285 ); rabbit polyclonal anti-MAP4K5 antibody (1:1,000, Cusabio, CSB-PA013440DSR2HU, RRID: AB_2892084 ); rabbit polyclonal anti-CREB1 antibody (1:1,000, Abclonal, A11989, RRID: AB_2758916 ); rabbit polyclonal anti-YAP1 antibody (1:1,000, Abclonal, A11430, RRID: AB_2758556 ); rabbit polyclonal anti-FYCO1 antibody (1:1,000, Cusabio, CSB-PA866262LA01HU, RRID: AB_2892085 ); rabbit polyclonal anti-FAF1 antibody (1:1,000, Abclonal, A2921, RRID: AB_2764739 ); goat polyclonal anti-Gal8 antibody (1:400, R&D Systems, AF1305, RRID: AB_2137229 ); rabbit polyclonal anti-KPNA3 (IMA4) antibody (1:1,000, Abclonal, A8347, RRID: AB_2770124 ); rabbit polyclonal anti-NUP107 antibody (1:1,000, Abclonal, A13110, RRID: AB_2759959 ); mouse monoclonal anti-IRS2 antibody (1:300, R&D Systems, MAB6347, 676415, RRID: AB_10992928 ); mouse monoclonal anti-FLAG M2 antibody (1:10,000, Sigma, F3165, RRID: AB_259529 ); mouse monoclonal anti-β-tubulin antibody (1:2000, AbLab, 21-0018-00, clone BT7R); mouse monoclonal anti-β-actin antibody (1:1,000, Abcam, ab8226, RRID: AB_306371 ); rabbit monoclonal anti-β-actin antibody (1:200, Abcam, ab115777, RRID: AB_10899528 ).

Techniques: Sequencing, Tandem Mass Spectroscopy, SDS Page, Western Blot, Incubation, Activity Assay, Infection

3CL pro disrupts galectin-8 binding to Spike in antiviral-autophagy (A) Immunoblot of human galectin-8 (Gal8) in BEAS-2B cells in response to IFN- α , IFN-β, or vehicle. One way ANOVA and Dunnett's posthoc test (mean ± SD, n = 3 each, *** p ≤ 0.001, * p ≤ 0.05). (B) MALDI-TOF-MS of intact versus 3CL pro -cleaved synthetic Gal8 P4–P4’ peptide. (C) SDS-PAGE and Edman sequencing of Gal8 incubated with 3CL pro +/− inhibitor GC376, or 3CL pro C145A (1:5 mol/mol, E:S). (D) Structural model of Gal8 docked onto 3CL pro . 3CL pro cleavage site identified by the neo-N-terminal peptide (red) in 9/9 independent TAILS analyses. (E) Gal8 immunoblots of lysates from primary HAECs incubated with 3CL pro or 3CL pro -C145A (1:200 w/w, E:S) for 18 h, 37°C (N = 5). (F) Gal8 immunoblot of infected Calu-3 cells at 24 (n = 4) and 48 (n = 4) hpi (MOI 1.0, mock n = 3). β-actin and β-tubulin loading controls. (G) ELISA of SARS-CoV-2 Spike S1 protein binding intact Gal8 or 3CL pro -cleaved (ΔGal8) (mean ± SD, n = 2, N = 2, ∗∗∗∗ p ≤ 0.0001, ∗∗ p ≤ 0.01, ns p > 0.05, two-way ANOVA with Šídák’s multiple comparison test). (H) Immunoprecipitation (IP) by α-FLAG agarose-beads of HeLa cell lysates co-transfected with GFP-NDP52, WT-Gal8-FLAG or C-Gal8 (159-317)-FLAG. (I) Intact or cleaved Gal8 binding immobilized NDP52 detected with anti-C-Gal8 antibody. Student's t test, (mean ± SD, n = 3, N = 2, ns p > 0.05). (J) NDP52 binding Spike S1-associated intact or cleaved ΔGal8 (mean ± SD, n = 2, N = 2, ∗∗∗∗ p ≤ 0.0001, ∗∗∗ p ≤ 0.001, ∗ p ≤ 0.05, ns p > 0.05, two-way ANOVA with Šídák’s multiple comparison test). (K) Confocal microscopy of NDP52 and FLAG immunofluorescence after osmotic shock of HEK293 cells transfected with FLAG-tagged (WT) Gal8, N-Gal8 (1-158), or C-Gal8 (159-317). Scale bar, 20 μm. Quantification of NDP52 and Gal8-positive puncta, mean ± SD, n = 30, N = 5, ∗∗∗∗ p ≤ 0.0001, ns p > 0.05, one-way ANOVA with Tukey’s multiple comparison test. (L) Human lung tissue sections from normal subjects (N = 3) and post-mortem COVID-19 patients (N = 4) stained with hematoxylin and eosin (H&E), DAPI (blue) and immunofluorescence on the same sections for Gal8 (green), NDP52 (red), Spike (magenta) and merged image (orange). Scale bar, 50 μm. (M) Numbers of immunopositive cells for Gal8, NDP52 and Gal8 colocalized with NDP52 in healthy versus COVID-19 lung tissue. Total cell count distribution shown as a scatter dot plot, bar = median. See also <xref ref-type=Figure S6 . " width="100%" height="100%">

Journal: Cell Reports

Article Title: Mechanistic insights into COVID-19 by global analysis of the SARS-CoV-2 3CL pro substrate degradome

doi: 10.1016/j.celrep.2021.109892

Figure Lengend Snippet: 3CL pro disrupts galectin-8 binding to Spike in antiviral-autophagy (A) Immunoblot of human galectin-8 (Gal8) in BEAS-2B cells in response to IFN- α , IFN-β, or vehicle. One way ANOVA and Dunnett's posthoc test (mean ± SD, n = 3 each, *** p ≤ 0.001, * p ≤ 0.05). (B) MALDI-TOF-MS of intact versus 3CL pro -cleaved synthetic Gal8 P4–P4’ peptide. (C) SDS-PAGE and Edman sequencing of Gal8 incubated with 3CL pro +/− inhibitor GC376, or 3CL pro C145A (1:5 mol/mol, E:S). (D) Structural model of Gal8 docked onto 3CL pro . 3CL pro cleavage site identified by the neo-N-terminal peptide (red) in 9/9 independent TAILS analyses. (E) Gal8 immunoblots of lysates from primary HAECs incubated with 3CL pro or 3CL pro -C145A (1:200 w/w, E:S) for 18 h, 37°C (N = 5). (F) Gal8 immunoblot of infected Calu-3 cells at 24 (n = 4) and 48 (n = 4) hpi (MOI 1.0, mock n = 3). β-actin and β-tubulin loading controls. (G) ELISA of SARS-CoV-2 Spike S1 protein binding intact Gal8 or 3CL pro -cleaved (ΔGal8) (mean ± SD, n = 2, N = 2, ∗∗∗∗ p ≤ 0.0001, ∗∗ p ≤ 0.01, ns p > 0.05, two-way ANOVA with Šídák’s multiple comparison test). (H) Immunoprecipitation (IP) by α-FLAG agarose-beads of HeLa cell lysates co-transfected with GFP-NDP52, WT-Gal8-FLAG or C-Gal8 (159-317)-FLAG. (I) Intact or cleaved Gal8 binding immobilized NDP52 detected with anti-C-Gal8 antibody. Student's t test, (mean ± SD, n = 3, N = 2, ns p > 0.05). (J) NDP52 binding Spike S1-associated intact or cleaved ΔGal8 (mean ± SD, n = 2, N = 2, ∗∗∗∗ p ≤ 0.0001, ∗∗∗ p ≤ 0.001, ∗ p ≤ 0.05, ns p > 0.05, two-way ANOVA with Šídák’s multiple comparison test). (K) Confocal microscopy of NDP52 and FLAG immunofluorescence after osmotic shock of HEK293 cells transfected with FLAG-tagged (WT) Gal8, N-Gal8 (1-158), or C-Gal8 (159-317). Scale bar, 20 μm. Quantification of NDP52 and Gal8-positive puncta, mean ± SD, n = 30, N = 5, ∗∗∗∗ p ≤ 0.0001, ns p > 0.05, one-way ANOVA with Tukey’s multiple comparison test. (L) Human lung tissue sections from normal subjects (N = 3) and post-mortem COVID-19 patients (N = 4) stained with hematoxylin and eosin (H&E), DAPI (blue) and immunofluorescence on the same sections for Gal8 (green), NDP52 (red), Spike (magenta) and merged image (orange). Scale bar, 50 μm. (M) Numbers of immunopositive cells for Gal8, NDP52 and Gal8 colocalized with NDP52 in healthy versus COVID-19 lung tissue. Total cell count distribution shown as a scatter dot plot, bar = median. See also Figure S6 .

Article Snippet: The primary antibodies and dilutions used were: mouse monoclonal anti-SARS-CoV-2 nucleocapsid antibody (1:1,000, Invitrogen, MA5-29981, RRID: AB_2785780 ); rabbit anti-SARS-CoV-1 3CL pro antibody (1:2000, Rockland, 200-401-A51, RRID: AB_828457 ); rabbit polyclonal anti-RPAP1 antibody (1:1,000, Proteintech, 15138-1-AP, RRID: AB_2301137 ); mouse monoclonal anti-PTBP1 antibody (1:500, Biolegend, 630101, 3H7, RRID: AB_2171285 ); rabbit polyclonal anti-MAP4K5 antibody (1:1,000, Cusabio, CSB-PA013440DSR2HU, RRID: AB_2892084 ); rabbit polyclonal anti-CREB1 antibody (1:1,000, Abclonal, A11989, RRID: AB_2758916 ); rabbit polyclonal anti-YAP1 antibody (1:1,000, Abclonal, A11430, RRID: AB_2758556 ); rabbit polyclonal anti-FYCO1 antibody (1:1,000, Cusabio, CSB-PA866262LA01HU, RRID: AB_2892085 ); rabbit polyclonal anti-FAF1 antibody (1:1,000, Abclonal, A2921, RRID: AB_2764739 ); goat polyclonal anti-Gal8 antibody (1:400, R&D Systems, AF1305, RRID: AB_2137229 ); rabbit polyclonal anti-KPNA3 (IMA4) antibody (1:1,000, Abclonal, A8347, RRID: AB_2770124 ); rabbit polyclonal anti-NUP107 antibody (1:1,000, Abclonal, A13110, RRID: AB_2759959 ); mouse monoclonal anti-IRS2 antibody (1:300, R&D Systems, MAB6347, 676415, RRID: AB_10992928 ); mouse monoclonal anti-FLAG M2 antibody (1:10,000, Sigma, F3165, RRID: AB_259529 ); mouse monoclonal anti-β-tubulin antibody (1:2000, AbLab, 21-0018-00, clone BT7R); mouse monoclonal anti-β-actin antibody (1:1,000, Abcam, ab8226, RRID: AB_306371 ); rabbit monoclonal anti-β-actin antibody (1:200, Abcam, ab115777, RRID: AB_10899528 ).

Techniques: Binding Assay, Western Blot, SDS Page, Sequencing, Incubation, Infection, Enzyme-linked Immunosorbent Assay, Protein Binding, Immunoprecipitation, Transfection, Confocal Microscopy, Immunofluorescence, Staining, Cell Counting

Journal: Cell Reports

Article Title: Mechanistic insights into COVID-19 by global analysis of the SARS-CoV-2 3CL pro substrate degradome

doi: 10.1016/j.celrep.2021.109892

Figure Lengend Snippet:

Article Snippet: The primary antibodies and dilutions used were: mouse monoclonal anti-SARS-CoV-2 nucleocapsid antibody (1:1,000, Invitrogen, MA5-29981, RRID: AB_2785780 ); rabbit anti-SARS-CoV-1 3CL pro antibody (1:2000, Rockland, 200-401-A51, RRID: AB_828457 ); rabbit polyclonal anti-RPAP1 antibody (1:1,000, Proteintech, 15138-1-AP, RRID: AB_2301137 ); mouse monoclonal anti-PTBP1 antibody (1:500, Biolegend, 630101, 3H7, RRID: AB_2171285 ); rabbit polyclonal anti-MAP4K5 antibody (1:1,000, Cusabio, CSB-PA013440DSR2HU, RRID: AB_2892084 ); rabbit polyclonal anti-CREB1 antibody (1:1,000, Abclonal, A11989, RRID: AB_2758916 ); rabbit polyclonal anti-YAP1 antibody (1:1,000, Abclonal, A11430, RRID: AB_2758556 ); rabbit polyclonal anti-FYCO1 antibody (1:1,000, Cusabio, CSB-PA866262LA01HU, RRID: AB_2892085 ); rabbit polyclonal anti-FAF1 antibody (1:1,000, Abclonal, A2921, RRID: AB_2764739 ); goat polyclonal anti-Gal8 antibody (1:400, R&D Systems, AF1305, RRID: AB_2137229 ); rabbit polyclonal anti-KPNA3 (IMA4) antibody (1:1,000, Abclonal, A8347, RRID: AB_2770124 ); rabbit polyclonal anti-NUP107 antibody (1:1,000, Abclonal, A13110, RRID: AB_2759959 ); mouse monoclonal anti-IRS2 antibody (1:300, R&D Systems, MAB6347, 676415, RRID: AB_10992928 ); mouse monoclonal anti-FLAG M2 antibody (1:10,000, Sigma, F3165, RRID: AB_259529 ); mouse monoclonal anti-β-tubulin antibody (1:2000, AbLab, 21-0018-00, clone BT7R); mouse monoclonal anti-β-actin antibody (1:1,000, Abcam, ab8226, RRID: AB_306371 ); rabbit monoclonal anti-β-actin antibody (1:200, Abcam, ab115777, RRID: AB_10899528 ).

Techniques: Recombinant, Sequencing, Fluorescence, Modification, Protease Inhibitor, Staining, Blocking Assay, Binding Assay, Activity Assay, Western Blot, Synthesized, Software, Microscopy, Spectrophotometry, Mass Spectrometry