cell signaling rabbit p samd2 18338 cst rabbit p smad3 9520 cst rabbit smad2  (Cell Signaling Technology Inc)

Journal: Cell Death and Differentiation

Article Title: PLK1/vimentin signaling facilitates immune escape by recruiting Smad2/3 to PD-L1 promoter in metastatic lung adenocarcinoma

doi: 10.1038/s41418-021-00781-4

Figure Lengend Snippet: a Analysis of transcriptome data for gene probes with fold changes of more than 1.5 (general cut-off point for DEGs) and a combined score calculated by multiplying the ln ( p value) and z -score among the invasive cells expressing active PLK1 during EMT of A549 cells. The significant genes were categorized using the KEGG 2019 pathway. b Analysis of the transcriptome data for gene probes in the invasive cells expressing active PLK1 during EMT of A549 cells. The network of ECM factors was extracted from the GeneMANIA database. c The overall survival (OS) times (left panel, n = 660) or the relapse free progression (RFP) rates (right panel, n = 383) of patients with lung adenocarcinoma were analysed according to their PLK1 and VIM expression levels. High (Hi) vs . low (Lo) expression was split by auto cut off value of KM PLOTTER. y; years. d Cumulative OS times of lung adenocarcinoma patients with stage 1 (left panel, n =360), or stage 2 (right panel, n = 132) were generated by splitting patients according to their expression of PLK1 and VIM . High (Hi) vs . low (Lo) expression was split by mean value. n.s., not significant. e A heatmap analysis was performed for VIM, PLK1 , epithelial markers CDH1 and OCLN , and several mesenchymal markers, including CDH2 , using a published transcriptome of TGF-β-treated NSCLC (GSE114761). f QRT-PCR was performed for CDH1, CDH2 , VIM, PLK1 , SNAI1 , and SNAI2 expression using A549 (left panel) and NCI-H460 (right panel) lung adenocarcinoma cells treated with 2.5 ng/ml of TGF-β for 48 hours. * p < 0.05; ** p < 0.01; *** p < 0.001; ( n = 3). Data are presented as mean ± SD. g Immunoblotting was performed to measure the expression and phosphorylation of PLK1 using anti-PLK1 and anti-p-PLK1 (T210) antibodies in A549 and NCI-H460 cells. The band intensity values of E-cadherin, N-cadherin, vimentin, p-Samd2 S465/S467 , p-PLK1 T210 , and PLK1 were quantified using LI-COR Odyssey software (Li-COR Biosciences). h The relative intensity values of p-T210-PLK1 and vimentin were normalized to those of PLK1 and GAPDH, respectively. All experiments were performed at least three independent experiments.

Article Snippet: After adjusting the protein concentration, proteins were resolved by SDS-PAGE and subjected to immunoblot analysis with the appropriate antibodies as follows: vimentin (Santa Cruz Biotechnology, sc-7557); phospho-vimentin S82 (MBL, D095-3); PLK1 (Millipore, 05-844); phospho-PLK1 T210 (Cell Signaling, 5472); N-cadherin (Sigma, C3865); E-cadherin (Cell Signaling, #4065); PD-L1 (Cell Signaling, 13684); PD-L2 (Invitrogen, PA5-20344); Smad2/3 (Cell Signaling, 8685); phospho-Samd2 S465/S467 (Cell Signaling, 18338); Stat3 (Santa Cruz Biotechnology, sc-8019); phospho-Stat3 T705 (Santa Cruz Biotechnology, sc-7993); c-Jun (Santa Cruz Biotechnology, sc-74543), c-fos (Santa Cruz Biotechnology, sc-52), NF-kB p65 (Santa Cruz Biotechnology, sc-71677), Erk1/2 (Cell Signaling, 4695), phospho-Erk1/2 T202/Y204 (Cell Signaling, 4370), Histone H1 (Santa Cruz Biotechnology, sc-8030), GAPDH (Sigma, G8795), β-actin (Sigma, A5441); and IgG (Santa Cruz Biotechnology, sc-2027).

Techniques: Expressing, Generated, Quantitative RT-PCR, Western Blot, Phospho-proteomics, Software

Journal: The Journal of Biological Chemistry

Article Title: Binding of Gtf2i-β/δ transcription factors to the ARMS2 gene leads to increased circulating HTRA1 in AMD patients and in vitro

doi: 10.1016/j.jbc.2021.100456

Figure Lengend Snippet: Action of HTRA1 on TGF-β/ALK5/SMAD2/3 signaling. A , we assayed the effects of HTRA1 on pSMAD2/3, SMAD2/2, VEGF, and TGFβRII by transfecting pCMV-Myc-HTRA1 vector in HEK-293, 661W and HeLa cells, followed by WB. Addition of the HTRA1 expression vector significantly inhibited SAMD2/3 phosphorylation but not SMAD 2/3 expression in HEK-293 cells. HTRA1 enhanced VEGF expression in HeLa cells and cleavage TGFβRII in all three cell lines. B , the band intensity was analyzed by Image J software. C , influence of overexpressed HTRA1 in mice. Phospho-Smad2/3 and TGFβRII, but not Smad2/3, decreased in 1-year-old Htra1 Tg mouse compared with WT mouse. VEGF enhanced in Htra1 Tg mouse compared with WT mouse. D , analysis of band intensities. E , mRNA levels of TGF II, TGFβRII, and ALK5 in HTRA1 transfected HEK-293, 661W, and HeLa cells, respectively. qRT-PCR analysis of TGF II, TGFβRII, and Alk5 mRNA levels ( F ) and VEGF isoforms (VEGF 120 , VEGF 164 , and VEGF 188 ) ( G ) in the retina of Htra1 Tg mouse. The expression of VEGF 120 isoform mRNA was significantly enhanced in 1-year-old Htra1 Tg mouse compared with WT. The other two VEGF isoforms were undetectable. Throughout, the results are expressed as the mean ± SEM. The p value was obtained by Student's t test.

Article Snippet: The primary antibodies used in WB included: TFII-I antibody (1:1000; CST; #4562), anti-hnRNP K antibody (1:10,000; Abcam; ab52600), EF-1r polyclonal antibody (1:1000; SAB; #40863), Lamin A/C (4c11) antibody (1:2000; CST; #4777); anti-actin (1/1000; Millipore; #MAB1501), ANTI-FLAG M2 (1:1000; SIGMA; F1804), anti-firefly luciferase antibody (1:1000; Abcam, ab21176), Samd2/3(D7F7) (1:1000; CST; #8685), Phospho-Samd2 (ser465/467)/smad3 (ser423/425) (D27F4) (1:1000; CST; #8828), anti-VEGF (1:1000; Abcam; ab46154), anti-HTRA1(1:500; R&D, MAB2916), anti-TGFβ RII(D-2) (1:300S; CB; sc-17799).

Techniques: Plasmid Preparation, Expressing, Phospho-proteomics, Software, Transfection, Quantitative RT-PCR