Journal: bioRxiv

Article Title: Extracellular Pgk1 enhances neurite outgrowth of motoneurons through Nogo66-independent targeting of NogoA

doi: 10.1101/550921

Figure Lengend Snippet: The molecular pathway mediated by extracellular addition of Pgk1 to enhance neurite outgrowth is independent from the neuronal Nogo66/NgR Pathway. ( A ) Western blot analysis. Protein levels of phosphorylated ROCK2 at Y256 (p-ROCK2-Y256), ROCK2, phosphorylated LimK1 at T508 (p-Limk1-T508), LimK1, phosphorylated Cofilin at S3 (p-Cofilin-S3), total Cofilin, phosphorylated EGFR at Y1173 (p-EGFR-Y1173), EGFR, phosphorylated Akt at S473 (p-Akt-S473) and Akt in neurons cultured with Sol8-vector CM, Sol8-NogoA CM and Sol8-NogoA CM plus Pgk1 addition were all examined, as indicated. The α–tubulin served as internal loading control. ( B-F ) Quantification of protein expression level. The band intensities shown on Western blotting were quantified by ImageJ software. The relative value of each examined protein was used for comparison among the three groups when the value obtained from the Sol8-vector CM group was set as 1. All of the above data were averaged from three independent experiments. Statistical analysis used Student’s t -test (***, p <0.001; **, p <0.01; *, p <0.05).

Article Snippet: The antibodies against NogoA (SC; 1:500), Cofilin (Cell Signaling; CS; 1:1000), phosphorylated Cofilin at S3 (CS; 1:1000), Rho-associated protein kinase 2 (ROCK2) (CS; 1:2000), phosphorylated ROCK2 at Y256 (Rockland, 1:2000), Epidermal growth factor receptor (EGFR) (SC; 1:500), phosphorylated EGFR at Y1173 (CS; 1:2000), Akt (Protein kinase B; PKB) (CS; 1:1000), phosphorylated Akt at S473 (CS; 1:1000), LIM domain kinase 1 (Limk1; SC; 1:500), phosphorylated Limk1 at S323 (Signalway Antibody; SAB; 1:1000), phosphorylated Limk1 at T508 (SAB; 1:500), Phosphoglycerate kinase 1 (Pgk1; Abcam; 1:2000), p21-activated kinase 1 (Pak1) (CS; 1:1000), phosphorylated Pak1 at T423 (CS; 1:1000), P38 mitogen-activated protein kinases (P38) (CS; 1:1000), phosphorylated P38 at T180 (CS; 1:1000), MAP kinase-activated protein kinase 2 (MK2) (CS; 1:1000), phosphorylated MK2 at T334 (CS; 1:1000), Synapsin I (Syn1) (Abcam; 1:1000), Growth Associated Protein 43 (GAP43) (Abcam; 1:1000), Choline acetyltransferase (ChAT) (Abcam; 1:1000), Microtubule-associated protein 2 (MAP2) (Abcam; 1:1000), α-tubulin (SA; 1:5000), Myc (Sigma; 1:2000), Flag (Abcam; 1:5000), mouse-HRP (SC; 1:5000) and rabbit-HRP (SC; 1:5000) were used.

Techniques: Western Blot, Cell Culture, Plasmid Preparation, Expressing, Software

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: Phospho-proteomics, Western Blot, Immunofluorescence, Staining

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

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, Control, shRNA, In Vitro, Recombinant, Incubation, Plasmid Preparation

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

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

Journal: The Kaohsiung journal of medical sciences

Article Title: The role of KDM4A-mediated histone methylation on temozolomide resistance in glioma cells through the HUWE1/ROCK2 axis.

doi: 10.1002/kjm2.12768

Figure Lengend Snippet: FIGURE 3 KDM4A regulation of ROCK2 and HUWE1 expression through histone demethylation. (A) Enrichment of KDM4A, H3K9me3, and H3K36me3 on ROCK2 and HUWE1 promoters assessed using ChIP. (B) Transcriptional levels of ROCK2 and HUWE1 evaluated by qRT-PCR. (C) Protein expressions of ROCK2 and HUWE1 analyzed by Western blot. Each experiment was independently repeated three times. Data in panels (A–C) were analyzed using two-way ANOVA, followed by Sidak's multiple comparisons test, *p < 0.05; **p < 0.01.

Article Snippet: The antibodies used included ROCK2 (1:1000, NB100-593, NOVUS) and HUWE1 (1:1000, ab70161, Abcam).

Techniques: Expressing, Quantitative RT-PCR, Western Blot

Journal: The Kaohsiung journal of medical sciences

Article Title: The role of KDM4A-mediated histone methylation on temozolomide resistance in glioma cells through the HUWE1/ROCK2 axis.

doi: 10.1002/kjm2.12768

Figure Lengend Snippet: FIGURE 4 HUWE1 binding to ROCK2 and promotion of its ubiquitination degradation. (A) Binding relationship between HUWE1 and ROCK2 assessed by co-immunoprecipitation, with IgG serving as the isotype control. (B) ROCK2 ubiquitination level in U251MG and T98G cells evaluated by co- immunoprecipitation and Western blot, with IgG serving as the isotype control. (C) ROCK2 ubiquitination level detected after adding MG132 (DMSO treatment as control) to different transfected cells, with IgG serving as the isotype control. Each experiment was independently repeated three times.

Article Snippet: The antibodies used included ROCK2 (1:1000, NB100-593, NOVUS) and HUWE1 (1:1000, ab70161, Abcam).

Techniques: Binding Assay, Ubiquitin Proteomics, Immunoprecipitation, Control, Western Blot, Transfection

Journal: The Kaohsiung journal of medical sciences

Article Title: The role of KDM4A-mediated histone methylation on temozolomide resistance in glioma cells through the HUWE1/ROCK2 axis.

doi: 10.1002/kjm2.12768

Figure Lengend Snippet: FIGURE 7 Chinese glioma genome atlas database analysis of ROCK2 and HUWE1 in GBM. (A–D) ROCK2 and HUWE1 expressions in different WHO grades of GBM and recurrent or primary GBM. (E and F) Survival analysis of GBM patients with different ROCK2 and HUWE1 expressions.

Article Snippet: The antibodies used included ROCK2 (1:1000, NB100-593, NOVUS) and HUWE1 (1:1000, ab70161, Abcam).

Techniques:

Journal: The Kaohsiung journal of medical sciences

Article Title: The role of KDM4A-mediated histone methylation on temozolomide resistance in glioma cells through the HUWE1/ROCK2 axis.

doi: 10.1002/kjm2.12768

Figure Lengend Snippet: FIGURE 8 Mechanism of KDM4A in glioma cell resistance to TMZ. In glioma cells, KDM4A reduces H3K9me3 to promote ROCK2 expression and reduces H3K36me3 to inhibit HUWE1 expression through histone demethylation. This represses HUWE1 binding to ROCK2 and inhibits ubiquitin degradation of ROCK2, ultimately promoting glioma cell resistance to TMZ.

Article Snippet: The antibodies used included ROCK2 (1:1000, NB100-593, NOVUS) and HUWE1 (1:1000, ab70161, Abcam).

Techniques: Expressing, Binding Assay, Ubiquitin Proteomics