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conditional rock2 k1007r  (Cyagen Biosciences)
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Cyagen Biosciences conditional rock2 k1007r
a Quantitative phosphoproteomic analysis in 16HBE cells treated with IL-13 (100 ng/ml) at 6 h post and during pretreatment with 2-D08 (30 μM). The bar graph shows the quantitative results of phosphorylated sites. b Enrichment and analysis of the protein domains of differentially expressed phosphorylated peptides. c The altered phosphorylation levels on the key phosphorylation sites of <t>ROCK2</t> upon IL-13 or/and 2-D08 treatment. d , e Western analyses in 16HBE cells treated with IL-13 at 6 h post and during treatment with 2-D08 ( n = 3, P values: 0.0002, 0.0015, 0.0016; <0.0001, 0.0076, 0.0003). f – m Lungs and bronchi from Fig. and Fig. were subjected to immunofluorescence staining and western analyses, respectively, each n = 6 or 3, P values: <0.0001, <0.0001, <0.0001 ( g ); 0.0015, 0.009, 0.0023 ( i ); <0.0001, <0.0001, <0.0001 ( k ); 0.0022, 0.0364, 0.0089 ( m ). Scale bar, 5 μm. Mean ± SD, One-way ANOVA and Tukey-Kramer multiple comparisons test, + P < 0.05, ++, ** P < 0.01. Source data are provided as a Source Data file.
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a Quantitative phosphoproteomic analysis in 16HBE cells treated with IL-13 (100 ng/ml) at 6 h post and during pretreatment with 2-D08 (30 μM). The bar graph shows the quantitative results of phosphorylated sites. b Enrichment and analysis of the protein domains of differentially expressed phosphorylated peptides. c The altered phosphorylation levels on the key phosphorylation sites of ROCK2 upon IL-13 or/and 2-D08 treatment. d , e Western analyses in 16HBE cells treated with IL-13 at 6 h post and during treatment with 2-D08 ( n = 3, P values: 0.0002, 0.0015, 0.0016; <0.0001, 0.0076, 0.0003). f – m Lungs and bronchi from Fig. and Fig. were subjected to immunofluorescence staining and western analyses, respectively, each n = 6 or 3, P values: <0.0001, <0.0001, <0.0001 ( g ); 0.0015, 0.009, 0.0023 ( i ); <0.0001, <0.0001, <0.0001 ( k ); 0.0022, 0.0364, 0.0089 ( m ). Scale bar, 5 μm. Mean ± SD, One-way ANOVA and Tukey-Kramer multiple comparisons test, + P < 0.05, ++, ** P < 0.01. Source data are provided as a Source Data file.

Journal: Nature Communications

Article Title: SUMOylation of Rho-associated protein kinase 2 induces goblet cell metaplasia in allergic airways

doi: 10.1038/s41467-023-39600-4

Figure Lengend Snippet: a Quantitative phosphoproteomic analysis in 16HBE cells treated with IL-13 (100 ng/ml) at 6 h post and during pretreatment with 2-D08 (30 μM). The bar graph shows the quantitative results of phosphorylated sites. b Enrichment and analysis of the protein domains of differentially expressed phosphorylated peptides. c The altered phosphorylation levels on the key phosphorylation sites of ROCK2 upon IL-13 or/and 2-D08 treatment. d , e Western analyses in 16HBE cells treated with IL-13 at 6 h post and during treatment with 2-D08 ( n = 3, P values: 0.0002, 0.0015, 0.0016; <0.0001, 0.0076, 0.0003). f – m Lungs and bronchi from Fig. and Fig. were subjected to immunofluorescence staining and western analyses, respectively, each n = 6 or 3, P values: <0.0001, <0.0001, <0.0001 ( g ); 0.0015, 0.009, 0.0023 ( i ); <0.0001, <0.0001, <0.0001 ( k ); 0.0022, 0.0364, 0.0089 ( m ). Scale bar, 5 μm. Mean ± SD, One-way ANOVA and Tukey-Kramer multiple comparisons test, + P < 0.05, ++, ** P < 0.01. Source data are provided as a Source Data file.

Article Snippet: The conditional caRhoA ( caRhoA +/− ) knock-in mouse strain with genetic background of C57BL/6J was generated by Cyagen Biosciences (Santa Clara, CA) as described previously , and the conditional Rock2 K1007R/+ knock-in founders with genetic background of C57BL/6 J were generated by CRISPR/Cas9 at Cyagen Biosciences as described previously .

Techniques: Western Blot, Immunofluorescence, Staining

a CC10-Cre and CC10-Cre; caRhoA +/- mice at 8 weeks of age were peritoneally injected with tamoxifen at 200 mg/kg on day 0, 1, 2, 3, and 4, and then intratracheally received 2-D08 at 10 or 30 mg/kg on day 9, 11, 13, and 15. Mice were euthanized on day 16 for the following analyses. b Cell counting and classification in BALFs. c – f H&E and PAS staining (scale bar, 10 μm), and immunostaining for Muc5AC and p-ROCK2 (scale bar, 5μm) in lung sections and their semi-quantification. P values: <0.0001, 0.0107, <0.0001 ( d ); <0.0001, 0.0134, <0.0001 ( e ); <0.0001, 0.0135, <0.0001 ( f ). g , h Western analyses and semi-quantification for bronchi ( P values: 0.0008, 0.0019, 0.0004). Mean ± SD, n = 6, One-way ANOVA and Tukey-Kramer multiple comparisons test, * , + P < 0.05, ** , ++ P < 0.01. Source data are provided as a Source Data file.

Journal: Nature Communications

Article Title: SUMOylation of Rho-associated protein kinase 2 induces goblet cell metaplasia in allergic airways

doi: 10.1038/s41467-023-39600-4

Figure Lengend Snippet: a CC10-Cre and CC10-Cre; caRhoA +/- mice at 8 weeks of age were peritoneally injected with tamoxifen at 200 mg/kg on day 0, 1, 2, 3, and 4, and then intratracheally received 2-D08 at 10 or 30 mg/kg on day 9, 11, 13, and 15. Mice were euthanized on day 16 for the following analyses. b Cell counting and classification in BALFs. c – f H&E and PAS staining (scale bar, 10 μm), and immunostaining for Muc5AC and p-ROCK2 (scale bar, 5μm) in lung sections and their semi-quantification. P values: <0.0001, 0.0107, <0.0001 ( d ); <0.0001, 0.0134, <0.0001 ( e ); <0.0001, 0.0135, <0.0001 ( f ). g , h Western analyses and semi-quantification for bronchi ( P values: 0.0008, 0.0019, 0.0004). Mean ± SD, n = 6, One-way ANOVA and Tukey-Kramer multiple comparisons test, * , + P < 0.05, ** , ++ P < 0.01. Source data are provided as a Source Data file.

Article Snippet: The conditional caRhoA ( caRhoA +/− ) knock-in mouse strain with genetic background of C57BL/6J was generated by Cyagen Biosciences (Santa Clara, CA) as described previously , and the conditional Rock2 K1007R/+ knock-in founders with genetic background of C57BL/6 J were generated by CRISPR/Cas9 at Cyagen Biosciences as described previously .

Techniques: Injection, Cell Counting, Staining, Immunostaining, Western Blot

a , b Western analyses in 293T cells transfected with Myc-SUMO1 for 24 h or the indicated times. c Co-immunoprecipitation experiments using a control IgG or a ROCK2 antibody in 16HBE cells and mouse primary bronchial epithelial cells (MPBEs). d Co-immunoprecipitation experiments using a Flag antibody in 16HBE cells transfected with Flag-ROCK2 in combination with Myc-SUMO1 in the presence of scramble or UBC9 shRNA. e In vitro SUMOylation assays in a reaction mixture containing ROCK2 recombinant protein, E1, E2, and SUMO1 and incubated at 37 °C or 4 °C for 60 min, followed by western analyses. f Western analyses in 293T cells 24 h after transfection with vector, wild-type (WT) ROCK2 or ROCK2 variants (K → R). g Co-immunoprecipitation experiments using a Flag antibody in 16HBE cells transfected with Myc-SUMO1 and Flag-ROCK2/Flag-ROCK2(K1007R). h Western analyses in 293T cells at 24 h post-transfection with or without Myc-SUMO1, UBC9 shRNA, and Flag-ROCK2 /Flag-ROCK2(K1007R). i In vitro SUMOylation assays in a reaction mixture containing ROCK2(WT) or ROCK2(K1007R) recombinant protein, E1, E2, and SUMO1, followed by western analyses. j , k Western analyses in 293 T cells transfected with Flag-ROCK2(WT or K1007R) in the presence of IL-13 stimulation for 6 h or of Myc-caRhoA. l Co-immunoprecipitation experiments using a Flag antibody in 16HBE cells transfected with HA-RhoA and Flag-ROCK2(WT or K1007R) after IL-13 treatment for 6 h. m 16HBE cells transfected with Flag-ROCK2(WT or K1007R), were subjected to GST pull-down assays with Rhotekin-RBD-coated beads, followed by western analyses. Experiments were repeated independently at least three times with similar results. Source data are provided as a Source Data file.

Journal: Nature Communications

Article Title: SUMOylation of Rho-associated protein kinase 2 induces goblet cell metaplasia in allergic airways

doi: 10.1038/s41467-023-39600-4

Figure Lengend Snippet: a , b Western analyses in 293T cells transfected with Myc-SUMO1 for 24 h or the indicated times. c Co-immunoprecipitation experiments using a control IgG or a ROCK2 antibody in 16HBE cells and mouse primary bronchial epithelial cells (MPBEs). d Co-immunoprecipitation experiments using a Flag antibody in 16HBE cells transfected with Flag-ROCK2 in combination with Myc-SUMO1 in the presence of scramble or UBC9 shRNA. e In vitro SUMOylation assays in a reaction mixture containing ROCK2 recombinant protein, E1, E2, and SUMO1 and incubated at 37 °C or 4 °C for 60 min, followed by western analyses. f Western analyses in 293T cells 24 h after transfection with vector, wild-type (WT) ROCK2 or ROCK2 variants (K → R). g Co-immunoprecipitation experiments using a Flag antibody in 16HBE cells transfected with Myc-SUMO1 and Flag-ROCK2/Flag-ROCK2(K1007R). h Western analyses in 293T cells at 24 h post-transfection with or without Myc-SUMO1, UBC9 shRNA, and Flag-ROCK2 /Flag-ROCK2(K1007R). i In vitro SUMOylation assays in a reaction mixture containing ROCK2(WT) or ROCK2(K1007R) recombinant protein, E1, E2, and SUMO1, followed by western analyses. j , k Western analyses in 293 T cells transfected with Flag-ROCK2(WT or K1007R) in the presence of IL-13 stimulation for 6 h or of Myc-caRhoA. l Co-immunoprecipitation experiments using a Flag antibody in 16HBE cells transfected with HA-RhoA and Flag-ROCK2(WT or K1007R) after IL-13 treatment for 6 h. m 16HBE cells transfected with Flag-ROCK2(WT or K1007R), were subjected to GST pull-down assays with Rhotekin-RBD-coated beads, followed by western analyses. Experiments were repeated independently at least three times with similar results. Source data are provided as a Source Data file.

Article Snippet: The conditional caRhoA ( caRhoA +/− ) knock-in mouse strain with genetic background of C57BL/6J was generated by Cyagen Biosciences (Santa Clara, CA) as described previously , and the conditional Rock2 K1007R/+ knock-in founders with genetic background of C57BL/6 J were generated by CRISPR/Cas9 at Cyagen Biosciences as described previously .

Techniques: Western Blot, Transfection, Immunoprecipitation, shRNA, In Vitro, Recombinant, Incubation, Plasmid Preparation

a , b Western analyses in 293T cells at 48 h post transfection with Myc-PIAS1, 2, 3, 4 or siRNAs of PIAS1, 2, 3, 4 in combination with or without Myc-SUMO1. c Western analyses in 293T cells at 48 h post transfection with or without Myc-PIAS1 and PIAS1 siRNA. d Co-immunoprecipitation experiments in 293T cells at 48 h post transfection with or without Myc-PIAS1 and Flag-ROCK2. e , f Western and co-immunoprecipitation analyses in 293T cells at 48 h post transfection with or without Myc-PIAS1 and Flag-ROCK2(WT or K1007R). g IHC of PIAS1 in human healthy bronchial sections ( n = 4, left: experimental group, right: negative control group, scale bar, 2 μm). h , i Immunostaining of CC10, PIAS1, DAPI and semi-quantification in BALF cells from children with FBA or asthma ( P value: <0.0001, scale bar, 10 μm). j – m Lungs or bronchi from NS- and OVA-challenged mice were subjected to IHC ( j ), qPCR ( k , P value: 0.0127), and western analyses ( l ) and their semi-quantification ( m , P value: 0.0093). Scale bar, 10 μm. n – q Mice were intratracheally instilled with lentiviral scramble- or PIAS1-shRNA and then with IL-13. Lungs and bronchi were subjected to H&E (scale bar, 10 μm), PAS (scale bar, 10 μm), Muc5AC (scale bar, 10 μm) and p-ROCK2 (scale bar, 5 μm) staining ( n , o , P values: <0.0001, <0.0001; <0.0001, <0.0001; <0.0001, 0.0014) and western analyses ( p , q , P values: <0.0001, 0.0002; 0.0002, 0.0031). Mean ± SD, n = 4, unpaired two-tailed Student’s t test or One-way ANOVA and Tukey-Kramer multiple comparisons test, * , + P < 0.05, ** , ++ P < 0.01. Experiments were repeated independently at least three times with similar results. Source data are provided as a Source Data file.

Journal: Nature Communications

Article Title: SUMOylation of Rho-associated protein kinase 2 induces goblet cell metaplasia in allergic airways

doi: 10.1038/s41467-023-39600-4

Figure Lengend Snippet: a , b Western analyses in 293T cells at 48 h post transfection with Myc-PIAS1, 2, 3, 4 or siRNAs of PIAS1, 2, 3, 4 in combination with or without Myc-SUMO1. c Western analyses in 293T cells at 48 h post transfection with or without Myc-PIAS1 and PIAS1 siRNA. d Co-immunoprecipitation experiments in 293T cells at 48 h post transfection with or without Myc-PIAS1 and Flag-ROCK2. e , f Western and co-immunoprecipitation analyses in 293T cells at 48 h post transfection with or without Myc-PIAS1 and Flag-ROCK2(WT or K1007R). g IHC of PIAS1 in human healthy bronchial sections ( n = 4, left: experimental group, right: negative control group, scale bar, 2 μm). h , i Immunostaining of CC10, PIAS1, DAPI and semi-quantification in BALF cells from children with FBA or asthma ( P value: <0.0001, scale bar, 10 μm). j – m Lungs or bronchi from NS- and OVA-challenged mice were subjected to IHC ( j ), qPCR ( k , P value: 0.0127), and western analyses ( l ) and their semi-quantification ( m , P value: 0.0093). Scale bar, 10 μm. n – q Mice were intratracheally instilled with lentiviral scramble- or PIAS1-shRNA and then with IL-13. Lungs and bronchi were subjected to H&E (scale bar, 10 μm), PAS (scale bar, 10 μm), Muc5AC (scale bar, 10 μm) and p-ROCK2 (scale bar, 5 μm) staining ( n , o , P values: <0.0001, <0.0001; <0.0001, <0.0001; <0.0001, 0.0014) and western analyses ( p , q , P values: <0.0001, 0.0002; 0.0002, 0.0031). Mean ± SD, n = 4, unpaired two-tailed Student’s t test or One-way ANOVA and Tukey-Kramer multiple comparisons test, * , + P < 0.05, ** , ++ P < 0.01. Experiments were repeated independently at least three times with similar results. Source data are provided as a Source Data file.

Article Snippet: The conditional caRhoA ( caRhoA +/− ) knock-in mouse strain with genetic background of C57BL/6J was generated by Cyagen Biosciences (Santa Clara, CA) as described previously , and the conditional Rock2 K1007R/+ knock-in founders with genetic background of C57BL/6 J were generated by CRISPR/Cas9 at Cyagen Biosciences as described previously .

Techniques: Western Blot, Transfection, Immunoprecipitation, Negative Control, Immunostaining, shRNA, Staining, Two Tailed Test

a , b Rock2 K1007R/K1007R , CC10-Cre; Rock2 K1007R/+ and CC10-Cre; Rock2 K1007R/K1007R mice were sensitized and challenged with OVA as described in Fig. , and bronchi were then subjected to western analyses and semi-quantification (each n = 3, P values: 0.0016, 0.0486, 0.0073). c , d Lungs (each n = 6) were subjected to paraffin-embedded sectioning, H&E and PAS staining, immunostaining for Muc5AC and p-ROCK2 ( c , scale bar, 10 μm), and their semi-quantification ( d , P values: <0.0001, 0.0001, <0.0001; <0.0001, 0.0115, <0.0001; <0.0001, <0.0001, <0.0001). e , f BALF cell counting and classification ( e , P values: <0.0001, <0.0001, <0.0001, <0.0001, <0.0001; 0.0006, 0.0437, 0.0159; <0.0001, <0.0001, 0.0006, 0.0155) and methacholine-provoked airway hyperreactivity ( f , each n = 6, P values: 0.0002, 0.0001, <0.0001, <0.0001, <0.0001; 0.049, 0.0399, 0.0073, 0.0095; 0.0196, 0.0019, 0.0035, 0.0002, 0.0003). g – l Rock2 K1007R/K1007R , CC10-Cre; Rock2 K1007R/+ and CC10-Cre; Rock2 K1007R/K1007R mice were intratracheally instilled with or without IL-13 as described in Fig. , and lungs or bronchi were subjected to H&E and PAS staining and immunostaining for Muc5AC and p-ROCK2 ( g – j , each n = 6, P values: <0.0001, 0.0001, <0.0001; <0.0001, 0.0036, <0.0001; <0.0001, <0.0001, <0.0001) or western analyses ( k , l , each n = 3, P values: 0.0015, 0.0236, 0.0034). Scale bar, 10 μm. Mean ± SD, One-way ANOVA and Tukey-Kramer multiple comparisons test, * , + P < 0.05, ** , ++ P < 0.01. Source data are provided as a Source Data file.

Journal: Nature Communications

Article Title: SUMOylation of Rho-associated protein kinase 2 induces goblet cell metaplasia in allergic airways

doi: 10.1038/s41467-023-39600-4

Figure Lengend Snippet: a , b Rock2 K1007R/K1007R , CC10-Cre; Rock2 K1007R/+ and CC10-Cre; Rock2 K1007R/K1007R mice were sensitized and challenged with OVA as described in Fig. , and bronchi were then subjected to western analyses and semi-quantification (each n = 3, P values: 0.0016, 0.0486, 0.0073). c , d Lungs (each n = 6) were subjected to paraffin-embedded sectioning, H&E and PAS staining, immunostaining for Muc5AC and p-ROCK2 ( c , scale bar, 10 μm), and their semi-quantification ( d , P values: <0.0001, 0.0001, <0.0001; <0.0001, 0.0115, <0.0001; <0.0001, <0.0001, <0.0001). e , f BALF cell counting and classification ( e , P values: <0.0001, <0.0001, <0.0001, <0.0001, <0.0001; 0.0006, 0.0437, 0.0159; <0.0001, <0.0001, 0.0006, 0.0155) and methacholine-provoked airway hyperreactivity ( f , each n = 6, P values: 0.0002, 0.0001, <0.0001, <0.0001, <0.0001; 0.049, 0.0399, 0.0073, 0.0095; 0.0196, 0.0019, 0.0035, 0.0002, 0.0003). g – l Rock2 K1007R/K1007R , CC10-Cre; Rock2 K1007R/+ and CC10-Cre; Rock2 K1007R/K1007R mice were intratracheally instilled with or without IL-13 as described in Fig. , and lungs or bronchi were subjected to H&E and PAS staining and immunostaining for Muc5AC and p-ROCK2 ( g – j , each n = 6, P values: <0.0001, 0.0001, <0.0001; <0.0001, 0.0036, <0.0001; <0.0001, <0.0001, <0.0001) or western analyses ( k , l , each n = 3, P values: 0.0015, 0.0236, 0.0034). Scale bar, 10 μm. Mean ± SD, One-way ANOVA and Tukey-Kramer multiple comparisons test, * , + P < 0.05, ** , ++ P < 0.01. Source data are provided as a Source Data file.

Article Snippet: The conditional caRhoA ( caRhoA +/− ) knock-in mouse strain with genetic background of C57BL/6J was generated by Cyagen Biosciences (Santa Clara, CA) as described previously , and the conditional Rock2 K1007R/+ knock-in founders with genetic background of C57BL/6 J were generated by CRISPR/Cas9 at Cyagen Biosciences as described previously .

Techniques: Western Blot, Staining, Immunostaining, Cell Counting