polyclonal antibodies against rhoa  (Cell Signaling Technology Inc)

Journal: Stem Cell Reviews and Reports

Article Title: HucMSCs-derived Exosomes Promote Lung Development in Premature Birth via Wnt5a/ROCK1 Axis

doi: 10.1007/s12015-024-10824-1

Figure Lengend Snippet: Term-Exos promoted the expression of lung development related proteins. A : WB examined the expressions of SPC and RhoA in E13.5 lung tissues. In term-Exos group, the expression of SPC and RhoA were obviously increasing. B : WB examined the expressions of SPC and RhoA in E18.5 lung tissues. Like which in E13.5 group, the expression of SPC and RhoA were improved in term-Exos groups. C : The expression of ABCA3 and SPB were examined by WB, which showed the expressions of ABCA3 in E13.5 and E18.5 stage were increasing after term-Exos injection, while the expressions of SPB precursor and mature SPB in E18.5 were obviously promoted after term-Exos injection. D , E , F : Real-time PCR examined the mRNA expressions of SFTPC, RhoA and ABCA3 in E13.5 and E18.5 lung tissue. Among E13.5 groups, the expression of ABCA3 mRNA was obviously increasing after term-Exos injection compared with the control while there were no significant differences in E18.5 ( n =3, *P<0.05, **P<0.01 ). Among E18.5 groups, the expression of SFTPC mRNA was increasing after term-Exos injection, while in E13.5, the result was opposite ( n =3, *P<0.05,**P<0.01 ). The expression of RhoA mRNA, however, was seemed to be inhibited after preterm-Exos use which was lower than that of term-Exos group, and there was no significant differences between the control and term-Exos group (n=3, *P<0.05,**P<0.01 )

Article Snippet: Overnight at 4 °C, the membranes were incubated with primary antibodies, which included monoclonal antibodies against SPC (Sigma, USA), ROCK1 (Cell Signaling Technology, USA) and p-ROCK1 (Invitrogen, USA), polyclonal antibodies against RhoA (Cell Signaling Technology, USA), Wnt5a (abcam, UK), LC3B (Cell Signaling Technology, USA), Atg5 (Abcam, UK), SPB (Santa cruz biotechnology, USA) and ABCA3 (Bioss, CHN), as well as a monoclonal antibody against β-actin (Millipore, US).

Techniques: Expressing, Injection, Real-time Polymerase Chain Reaction, Control

Journal: Stem Cell Reviews and Reports

Article Title: HucMSCs-derived Exosomes Promote Lung Development in Premature Birth via Wnt5a/ROCK1 Axis

doi: 10.1007/s12015-024-10824-1

Figure Lengend Snippet: A schematic model showing the proposed mechanism for the role of exosomal Wnt5a in lung development. Term-Exos: Term infant umbilical cord mesenchymal stem cells-derived exosomes, Ror: Receptor tyrosine kinase-like orphan receptor; FZD 6 : Frizzled receptor 6; ROCK1: Rho-associated protein kinase 1; RhoA: Ras homolog gene family, member A; LC3B: Microtubule-associated proteins 1A/1B light chain 3B; P: Phosphorylation; α-SMA: alpha smooth muscle actin

Article Snippet: Overnight at 4 °C, the membranes were incubated with primary antibodies, which included monoclonal antibodies against SPC (Sigma, USA), ROCK1 (Cell Signaling Technology, USA) and p-ROCK1 (Invitrogen, USA), polyclonal antibodies against RhoA (Cell Signaling Technology, USA), Wnt5a (abcam, UK), LC3B (Cell Signaling Technology, USA), Atg5 (Abcam, UK), SPB (Santa cruz biotechnology, USA) and ABCA3 (Bioss, CHN), as well as a monoclonal antibody against β-actin (Millipore, US).

Techniques: Derivative Assay, Phospho-proteomics

Journal: Journal of Cellular and Molecular Medicine

Article Title: Use of iTRAQ ‐based quantitative proteomic identification of CHGA and UCHL1 correlated with lymph node metastasis in colorectal carcinoma

doi: 10.1111/jcmm.17793

Figure Lengend Snippet: The effect of chromogranin A (CHGA) and ubiquitin carboxyl‐terminal hydrolase isozyme L1 (UCHL1) silencing on Rho‐GTPase/AKT/NFκB pathway activation and the expression of EMT markers in CRC. After transfection with lentiviral shRNA targeting CHGA and UCHL1 (shCHGA and shUCHL1) or non‐targeting control (shControl), whole‐cell lysate proteins of HCT‐116 cells were subjected to western blotting, with antibodies against phosphorylation of Rho‐GTPase, AKT and NFκB p50 (A) as well as β‐catenin, cyclin E, twist 1/2 and vimentin (B) β‐Actin served as the loading control. The protein levels were quantified through densitometric analysis with the ratio of the untreated control (Control) set as onefold. The quantitative data are presented as the mean of three repeats from three independent experiments. * p < 0.05, compared with the control group.

Article Snippet: The antibodies were respectively obtained by Abcam Technology (Abcam) and Cell Signalling Technology, including mouse/rabbit polyclonal antibodies against RhoA Ser188, AKT Thr180Tyr182 and NFκB p50 bought from all culture materials were obtained from Gibco (Grand Island, NY, USA).

Techniques: Ubiquitin Proteomics, Activation Assay, Expressing, Transfection, shRNA, Control, Western Blot, Phospho-proteomics

Journal: Journal of Cellular and Molecular Medicine

Article Title: Use of iTRAQ ‐based quantitative proteomic identification of CHGA and UCHL1 correlated with lymph node metastasis in colorectal carcinoma

doi: 10.1111/jcmm.17793

Figure Lengend Snippet: Schematic presentation of the working model of CHGA and UCHL1 participate in promotion of the LNM‐associated CRC through endogenous Rho‐GTPase/AKT/NFκB signalling pathways‐mediated histone modification (H3K4me3) as reliable candidate LNM‐associated markers.

Article Snippet: The antibodies were respectively obtained by Abcam Technology (Abcam) and Cell Signalling Technology, including mouse/rabbit polyclonal antibodies against RhoA Ser188, AKT Thr180Tyr182 and NFκB p50 bought from all culture materials were obtained from Gibco (Grand Island, NY, USA).

Techniques: Modification

Journal: Journal of Cellular and Molecular Medicine

Article Title: Use of iTRAQ ‐based quantitative proteomic identification of CHGA and UCHL1 correlated with lymph node metastasis in colorectal carcinoma

doi: 10.1111/jcmm.17793

Figure Lengend Snippet: Alternation of histone modification of CHGA and UCHL1 promoters by the Rho‐GTPase/AKT/NFκB signalling pathways. After incubation with various concentrations of the specific inhibitors such as CCG‐1423, PDTC and wortmannin for 24 h, chromatin immunoprecipitation (ChIP) assays using antibodies against H3K4me3 was performed to pull down associated DNA in the HCT‐116 cells. Polymerase chain reaction amplified the precipitated DNA by using primer sets specific to the target sites (−1185 to −940 and −407 to −230) of CHGA and UCHL1 promoters. After normalized signal to the negative control ChIP asΔCT by subtracting the mean CT of the input from that of the individual region among the untreated control group, using the ΔΔCt method to calculate the effect of the specific inhibitors on specific genes. The data are presented as fold change of the untreated group (mean ± SD) of three independent experiments with * p < 0.01.

Article Snippet: The antibodies were respectively obtained by Abcam Technology (Abcam) and Cell Signalling Technology, including mouse/rabbit polyclonal antibodies against RhoA Ser188, AKT Thr180Tyr182 and NFκB p50 bought from all culture materials were obtained from Gibco (Grand Island, NY, USA).

Techniques: Modification, Incubation, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Amplification, Negative Control, Control