rabbit anti α sma  (Bioss)

Journal: Frontiers in Immunology

Article Title: METTL3-driven m 6 A modification of NLRC5 promotes renal fibrosis in chronic kidney disease through Keap1/Nrf2/ARE signaling pathway

doi: 10.3389/fimmu.2026.1739011

Figure Lengend Snippet: METTL3 regulated m 6 A modification and expression of NLRC5 in TGF-β1-induced cell fibrosis model. (A) Quantitative reverse transcription PCR analysis of METTL3 mRNA levels in HK-2 cells transfected with OE-METTL3 or si-METTL3, followed by TGF-β1 (10 ng/mL) treatment for 48 h (*** p < 0.001, vs. Vector; ### p < 0.001, vs. si-NC). (B) Western blot confirming METTL3 protein expression in the same conditions as (A, C) Quantitative reverse transcription PCR showing NLRC5 mRNA levels after METTL3 overexpression or knockdown (** p < 0.01, *** p < 0.001, vs. vector or si-NC). (D) Western blot analysis of NLRC5 protein expression under the indicated conditions (*** p < 0.001, vs. vector; ### p < 0.001, vs. si-NC). (E) NLRC5 mRNA stability assessed by actinomycin D chase assay in METTL3-modulated cells, measured by quantitative reverse transcription PCR at 0, 4, and 8 h. (F) Predicted m6A methylation sites in the NLRC5 transcript analyzed by SRAMP. The graph illustrates the distribution of m6A sites across the transcript, with confidence levels indicated by different color thresholds. (G) MeRIP-qPCR showing reduced m 6 A enrichment on NLRC5 mRNA upon METTL3 knockdown. (H) RIP-qPCR analysis indicating direct interaction between METTL3 and NLRC5 mRNA, with IgG as control (** p < 0.01, *** p < 0.001, vs. indicated controls). (I) Western blot of fibrosis markers (α-SMA, Collagen I, and E-cadherin) following METTL3 overexpression or knockdown, with or without STM2457 treatment (*** p < 0.001, vs. vector; ### p < 0.001, vs. OE-METTL3; && p < 0.01, &&& p < 0.001, vs. si-NC). Data are presented as mean ± SD from three independent experiments.

Article Snippet: After blocking with 5% non-fat milk in TBST for 2 h at room temperature, membranes were incubated overnight at 4 °C with primary antibodies targeting α-SMA (1:1000, #19245), Collagen I (1:1000, #72026), E-cadherin (1:1000, #3195), Nrf2 (1:1000, #12721), Keap1 (1:1000, #8047), HO-1 (1:1000, #70081), NQO1 (1:1000, #62262), METTL3 (1:1000, #86132), NLRC5 (1:1000, #72379), Lamin B1 (1:1000, #13435), Lamin B1 (1:1000, #13435) and GAPDH (1:5000, #5174) (all from Cell Signaling Technology unless otherwise stated; dilution 1:1000, GAPDH 1:5000).

Techniques: Modification, Expressing, Reverse Transcription, Transfection, Plasmid Preparation, Western Blot, Over Expression, Knockdown, Methylation, Control

Journal: Frontiers in Immunology

Article Title: METTL3-driven m 6 A modification of NLRC5 promotes renal fibrosis in chronic kidney disease through Keap1/Nrf2/ARE signaling pathway

doi: 10.3389/fimmu.2026.1739011

Figure Lengend Snippet: Effects of NLRC5 knockdown on inflammation, oxidative stress, and fibrosis in TGF-β1–treated HK-2 cells. (A, B) Quantitative reverse transcription PCR (A) and Western blot analysis (B) confirming the efficient knockdown of NLRC5 expression in TGF-β1–stimulated HK-2 cells transfected with si-NLRC5 (*** p < 0.001, vs. si-NC). (C) ELISA detection of IL-1β and TNF-α levels in cell culture supernatants following TGF-β1 treatment and/or NLRC5 silencing. (D) Measurement of oxidative stress indicators, including MDA content and SOD activity, under different experimental conditions. (E) Representative fluorescence images and quantitative analysis of intracellular ROS levels detected using the DCFDA probe in the indicated groups. (F) Western blot analysis of fibrosis-related proteins, including α-SMA, Collagen I, and E-cadherin, in TGF-β1–treated HK-2 cells with or without NLRC5 knockdown (*** p < 0.001, vs. control; # p < 0.05, ## p < 0.01, ### p < 0.001, vs. TGF-β1 + si-NC). Data are presented as mean ± SD from three independent experiments.

Article Snippet: After blocking with 5% non-fat milk in TBST for 2 h at room temperature, membranes were incubated overnight at 4 °C with primary antibodies targeting α-SMA (1:1000, #19245), Collagen I (1:1000, #72026), E-cadherin (1:1000, #3195), Nrf2 (1:1000, #12721), Keap1 (1:1000, #8047), HO-1 (1:1000, #70081), NQO1 (1:1000, #62262), METTL3 (1:1000, #86132), NLRC5 (1:1000, #72379), Lamin B1 (1:1000, #13435), Lamin B1 (1:1000, #13435) and GAPDH (1:5000, #5174) (all from Cell Signaling Technology unless otherwise stated; dilution 1:1000, GAPDH 1:5000).

Techniques: Knockdown, Reverse Transcription, Western Blot, Expressing, Transfection, Enzyme-linked Immunosorbent Assay, Cell Culture, Activity Assay, Fluorescence, Control

Journal: Frontiers in Immunology

Article Title: METTL3-driven m 6 A modification of NLRC5 promotes renal fibrosis in chronic kidney disease through Keap1/Nrf2/ARE signaling pathway

doi: 10.3389/fimmu.2026.1739011

Figure Lengend Snippet: Involvement of the Keap1/Nrf2/ARE signaling pathway in the regulatory effects of NLRC5 knockdown in TGF-β1–induced HK-2 cells. (A) Western blot analysis of Keap1, nuclear Nrf2, and the Nrf2 downstream targets HO-1 and NQO-1 in HK-2 cells under the indicated conditions (Control, TGF-β1, TGF-β1 + si-NC, TGF-β1 + si-NLRC5, TGF-β1 + si-NLRC5 + OE-Keap1, and TGF-β1 + si-NLRC5 + ML385). Statistical significance is indicated as follows: *** p < 0.001, vs. control; # p < 0.05, ### p < 0.001, vs . TGF-β1 + si-NC; & p < 0.05, &&& p < 0.001, vs . TGF-β1 + si-NLRC5. (B) ELISA determination of IL-1β and TNF-α levels in cell culture supernatants following the indicated treatments (*** p < 0.001, vs. si-NC; # p < 0.05, ## p < 0.01, vs. si-NLRC5). (C) Quantitative analysis of malondialdehyde (MDA) content and superoxide dismutase (SOD) activity in different groups (*** p < 0.001, vs. si-NC; # p < 0.05, ## p < 0.01, vs. si-NLRC5). (D) Representative fluorescence images and quantitative measurement of intracellular reactive oxygen species (ROS) using the DCFDA probe (*** p < 0.001, vs. si-NC; ### p < 0.001, vs. si-NLRC5). (E) Western blot analysis of fibrosis-associated proteins, including α-SMA, Collagen I, and E-cadherin, in HK-2 cells under the indicated conditions (*** p < 0.001, vs. TGF-β1 + si-NC, ## p < 0.01, ### p < 0.001, vs. TGF-β1 + si-NLRC5). Data are presented as mean ± SD from three independent experiments.

Article Snippet: After blocking with 5% non-fat milk in TBST for 2 h at room temperature, membranes were incubated overnight at 4 °C with primary antibodies targeting α-SMA (1:1000, #19245), Collagen I (1:1000, #72026), E-cadherin (1:1000, #3195), Nrf2 (1:1000, #12721), Keap1 (1:1000, #8047), HO-1 (1:1000, #70081), NQO1 (1:1000, #62262), METTL3 (1:1000, #86132), NLRC5 (1:1000, #72379), Lamin B1 (1:1000, #13435), Lamin B1 (1:1000, #13435) and GAPDH (1:5000, #5174) (all from Cell Signaling Technology unless otherwise stated; dilution 1:1000, GAPDH 1:5000).

Techniques: Knockdown, Western Blot, Control, Enzyme-linked Immunosorbent Assay, Cell Culture, Activity Assay, Fluorescence

Journal: Frontiers in Immunology

Article Title: METTL3-driven m 6 A modification of NLRC5 promotes renal fibrosis in chronic kidney disease through Keap1/Nrf2/ARE signaling pathway

doi: 10.3389/fimmu.2026.1739011

Figure Lengend Snippet: Effects of METTL3 knockdown and NLRC5 modulation on Keap1/Nrf2/ARE signaling and fibrotic responses in TGF-β1–induced HK-2 cells. (A) Western blot analysis of Keap1, nuclear Nrf2, and Nrf2 downstream targets HO-1 and NQO-1 in TGF-β1–stimulated HK-2 cells transfected with si-NC, si-METTL3, si-METTL3 + ML385, or si-METTL3 + OE-NLRC5. (B) ELISA analysis of IL-1β and TNF-α levels in the culture supernatants of the indicated groups. (C) Quantitative assessment of oxidative stress markers, including MDA content and SOD activity, under the indicated treatments. (D) Representative fluorescence images and quantitative analysis of intracellular ROS levels detected by the DCFDA probe. (E) Western blot analysis of fibrosis-related proteins, including α-SMA, Collagen I, and E-cadherin, in TGF-β1–treated HK-2 cells transfected as indicated. Data are presented as mean ± SD from three independent experiments. *** p < 0.001, versus si-NC; # p < 0.05, ## p < 0.01, ### p < 0.001, versus si-METTL3.

Article Snippet: After blocking with 5% non-fat milk in TBST for 2 h at room temperature, membranes were incubated overnight at 4 °C with primary antibodies targeting α-SMA (1:1000, #19245), Collagen I (1:1000, #72026), E-cadherin (1:1000, #3195), Nrf2 (1:1000, #12721), Keap1 (1:1000, #8047), HO-1 (1:1000, #70081), NQO1 (1:1000, #62262), METTL3 (1:1000, #86132), NLRC5 (1:1000, #72379), Lamin B1 (1:1000, #13435), Lamin B1 (1:1000, #13435) and GAPDH (1:5000, #5174) (all from Cell Signaling Technology unless otherwise stated; dilution 1:1000, GAPDH 1:5000).

Techniques: Knockdown, Western Blot, Transfection, Enzyme-linked Immunosorbent Assay, Activity Assay, Fluorescence

Journal: Frontiers in Immunology

Article Title: METTL3-driven m 6 A modification of NLRC5 promotes renal fibrosis in chronic kidney disease through Keap1/Nrf2/ARE signaling pathway

doi: 10.3389/fimmu.2026.1739011

Figure Lengend Snippet: Effects of METTL3 knockdown on UUO-induced renal fibrosis, oxidative stress and the Keap1/Nrf2/ARE pathway in UUO mice. (A) Masson’s trichrome staining showing collagen deposition around renal tubules in different groups (n = 8) and quantitative analysis of fibrosis area. Scale bar = 100 μm. (B) Immunohistochemical staining of α-SMA in renal cortex tissues. Brown staining represents α-SMA–positive cells. Scale bar = 50 μm. Serum levels of oxidative stress markers, including MDA (C) and SOD (D) , in each group, assessed by biochemical assays. (E) Western blot analysis and quantification of Nrf2, Keap1, α-SMA, and Collagen I protein expression in kidney tissues. Data are presented as mean ± SD. Statistical analysis was performed using one-way ANOVA with Tukey’s test (n = 8 in each group). *** p < 0.001, versus sham; ## p < 0.01, ### p < 0.001, versus UUO + Ad-shNC.

Article Snippet: After blocking with 5% non-fat milk in TBST for 2 h at room temperature, membranes were incubated overnight at 4 °C with primary antibodies targeting α-SMA (1:1000, #19245), Collagen I (1:1000, #72026), E-cadherin (1:1000, #3195), Nrf2 (1:1000, #12721), Keap1 (1:1000, #8047), HO-1 (1:1000, #70081), NQO1 (1:1000, #62262), METTL3 (1:1000, #86132), NLRC5 (1:1000, #72379), Lamin B1 (1:1000, #13435), Lamin B1 (1:1000, #13435) and GAPDH (1:5000, #5174) (all from Cell Signaling Technology unless otherwise stated; dilution 1:1000, GAPDH 1:5000).

Techniques: Knockdown, Staining, Immunohistochemical staining, Western Blot, Expressing

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Fibroblast growth factor-inducible 14 accelerates pulmonary fibrosis by inducing fibroblast senescence in mice

doi: 10.1007/s00018-026-06161-w

Figure Lengend Snippet: Fn14 shRNA alleviates BLM-induced PF in mice. A , C57BL/6J mice received empty adenovirus or Fn14 vectors (5 × 10 8 PFU/20 g, i.t. ). Seven days later, mice were administered BLM (2.5 mg/kg, i.t. ) to establish a PF model. Mice were sacrificed after the BLM administration on the 21st day. B , the expression of Fn14 mRNA in the lungs was detected by Real-time PCR. C-D , the expression of Fn14 protein in the lungs was detected by Western blot ( n = 6–8). E, weight change of mice ( n = 6–10). F, the rate of weight change in mice was calculated. G, the pulmonary tissue histopathology of mice was stained with H&E (Top row), and the collagen deposition was detected by Masson’s trichrome staining (Bottom row). H, the Ashcroft score was evaluated by three blinded pathologists. I, the collagen subtype was detected by Sirius red staining. J, the expressions of Col1a1 , Col3a1 , and Acta2 mRNA in the lungs were detected by Real-time PCR. L, the expressions of Collagen I, Collagen III, and α-SMA proteins in the lungs were assayed by Western blot ( n = 6–8). ***P < 0.001

Article Snippet: Anti-α-SMA polyclonal antibody , CST , 19,245 , 1: 5000.

Techniques: shRNA, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Histopathology, Staining