rabbit anti kim1  (Novus Biologicals)

Journal: The FASEB Journal

Article Title: Regulators of calcineurin 1 deficiency attenuates tubulointerstitial fibrosis through improving mitochondrial fitness

doi: 10.1096/fj.202000781rrr

Figure Lengend Snippet: FIGURE 3 Generation of TEC-specific RCAN1 deletion mice. A, Schematic of RCAN1f/f mice generation by CRISPR/Cas9-stimulated homologous recombination. B, Mating strategy to generate RCAN1 conditional knockout in mouse TECs. C, Successful transmission of Cdh16-Cre and RCAN1f/f was confirmed by PCR genotyping. D, Reduced mRNA expression of RCAN1 in renal cortex of RCAN1tecKO mice was confirmed by quantitative RT-PCR (n = 3, *P < .05, **P < .01, ***P < .001). E, Representative images of kidney sections stained by in situ hybridization for RCAN1.1 and RCAN1.4 from sham-operated and obstructed kidneys of RCAN1f/f and RCAN1tecKO mice 7 d after UUO. F, G, The mRNA levels of Kim1 and Ngal were assayed by quantitative RT-PCR (n = 4, *P < .05, ***P < .001). H, The mean tubular damage scores were assessed using a light microscope (n = 5-6, ***P < .001). Ten randomly high-power fields were chosen in kidney sections after UUO. I, Representative micrographs of multiple immunofluorescence of RCAN1 and TEC markers AQP1 and AQP3 (200×) in sham operation or 7 days after UUO. Scale bars, 20 μm. J, Representative micrographs of HE-stained kidney sections of RCAN1f/f and RCAN1tecKO mice with sham operation or 7 days after UUO. Scale bars, 200 μm. K, Representative images of immunohistochemical staining for F4/80 (pan-macrophage marker)

Article Snippet: Primary antibodies for RCAN1 (D6694), FLAG (F3165), fibronectin (2716206), and LC3 (L8918) were from MilliporeSigma; antibodies for phospho-Smad3 (9520S), E-cadherin (3195S), α-SMA (19245S), Drp1 (8570S), phospho-Drp1 (Ser637, 4867S), phospho-Drp1 (Ser616, 3455S), Mff (84580S), Mfn1 (14739S), Mfn2 (9482S), Opa1 (80471S), Parkin (4211S), P62 (5114S), Kim1 (14971S), Bax (2772S), Bcl-2 (3498S), cytochrome C (4280S), caspase-3 (9662S), caspase-9 (9508S), cytochrome c oxidase IV (COX IV) (4844S), β-tubulin (2146S), β-actin (4970S), and GAPDH were from CST.

Techniques: CRISPR, Homologous Recombination, Knock-Out, Transmission Assay, Expressing, Quantitative RT-PCR, Staining, In Situ Hybridization, Light Microscopy, Immunofluorescence, Immunohistochemical staining, Marker

Journal: The FASEB Journal

Article Title: Regulators of calcineurin 1 deficiency attenuates tubulointerstitial fibrosis through improving mitochondrial fitness

doi: 10.1096/fj.202000781rrr

Figure Lengend Snippet: FIGURE 8 TEC-specific deletion of RCAN1 reduces tubular programed cell death after UUO. A, Representative micrographs of immunostaining of cleaved caspase-3 in kidney sections of RCAN1f/f and RCAN1tecKO mice with sham operation or 2 days after UUO and quantification was shown in right panel (n = 3, *P < .05 vs RCAN1f/f with sham operation, #P < .05 vs RCAN1f/f 2 days after UUO). Scale bars, 200 μm. B, Representative micrographs of TUNEL-stained kidney sections of RCAN1f/f and RCAN1tecKO mice with sham operation or 7 days after UUO. Cells undergoing apoptosis gave out green fluorescence. Scale bars, 200 μm. Quantification of TUNEL-positive cells was shown in right panel (n = 3). C, Western blot analysis of Kim1, RIPK1/3, Bax, Bcl-2, cytosol cytochrome C (CytC), cleaved caspase-9 (Cas-9), and Cas-3. (n = 5). *P < .05 vs RCAN1f/f with sham operation, #P < .05 vs RCAN1f/f 7 days after UUO (B, C). D, Primary cultured tubular cells from WT or RCAN1tecKO mice were incubated with 5 ng/mL recombinant human TGF-β1 for 24 hours, and then, stained with annexin V-FITC and PI to determine cell apoptosis using a flow cytometry assay (*P < .05 vs WT, n = 4). E, The protein levels of Bax, cleaved caspase-3, RIPK1, and RIPK3 were detected by Western blot at the same conditions described in D (*P < .05 vs WT, #P < .05 vs WT + TGF-β1)

Article Snippet: Primary antibodies for RCAN1 (D6694), FLAG (F3165), fibronectin (2716206), and LC3 (L8918) were from MilliporeSigma; antibodies for phospho-Smad3 (9520S), E-cadherin (3195S), α-SMA (19245S), Drp1 (8570S), phospho-Drp1 (Ser637, 4867S), phospho-Drp1 (Ser616, 3455S), Mff (84580S), Mfn1 (14739S), Mfn2 (9482S), Opa1 (80471S), Parkin (4211S), P62 (5114S), Kim1 (14971S), Bax (2772S), Bcl-2 (3498S), cytochrome C (4280S), caspase-3 (9662S), caspase-9 (9508S), cytochrome c oxidase IV (COX IV) (4844S), β-tubulin (2146S), β-actin (4970S), and GAPDH were from CST.

Techniques: Immunostaining, TUNEL Assay, Staining, Fluorescence, Western Blot, Cell Culture, Incubation, Recombinant, Flow Cytometry