144 envision system hrp labelled polymer anti rabbit dako k4003 tsa alexa fluor 488 tyramide  (Thermo Fisher)

Journal: The FASEB Journal

Article Title: Klotho regulation by albuminuria is dependent on ATF3 and endoplasmic reticulum stress

doi: 10.1096/fj.201900893r

Figure Lengend Snippet: FIGURE 5 Albumin induces ER stress in vitro and in vivo. Relative mRNA and protein levels of ER stress response genes as measured by qPCR (N = 10), (A) and western blot (N = 8), (B), respectively, in HEK-293 overexpressing cells exposed to 10 mg/mL BSA for 24 hours. Relative mRNA and protein levels of ER stress response genes as measured by qPCR (N = 10), (C) and western blot (N = 8), (D), respectively, in the HK-2 cells exposed to 10 mg/mL BSA for 24 hours. Relative mRNA levels of ER stress response genes in the cortex of control (N = 13) and proteinuric mice (N = 16) 7 days post-injection (E). Representative immunohistochemical staining of ATF4 and ATF3 transcription factors in kidney sections (F). The mRNA levels are normalized to RPLP0 gene expression and the protein levels to β-actin. Mean ± SD, multiple t test without correction *P < .05

Article Snippet: To isolate the lentiviral particles, the cell medium was collected 48 hours after and centrifuged at 1500 rpm for 7 minutes and passed through a Name Host and isotype Manufacturer Cat. No. Dilution Primary antibody Klotho Rat IgG2a TransGenic KO603 1:50 ATF4 Rabbit IgG Cell signaling #11815 1:250 ATF3 Rabbit IgG Abcam ab207434 1:50 Detection system Klotho ImmPRESS HRP AntiRat IgG Vector Laboratories MP-7404-50 EnVision Flex kit DAKO K8010 ATF4, ATF3 CSA II BiotinFree Catalyzed Amplification System DAKO K149711-2 T A B L E 1 Antibodies and kits used for immunohistochemistry | 5DELITSIKOU ET aL. 0.45-μM pore size filter to remove cell debris.

Techniques: In Vitro, In Vivo, Western Blot, Control, Injection, Immunohistochemical staining, Staining, Gene Expression

Journal: The FASEB Journal

Article Title: Klotho regulation by albuminuria is dependent on ATF3 and endoplasmic reticulum stress

doi: 10.1096/fj.201900893r

Figure Lengend Snippet: FIGURE 6 ATF4 and ATF3 regulate Klotho expression. Relative mRNA and protein levels of Klotho, ATF4 and ATF3 as measured by qPCR and western blot, respectively, in HK-2 cells transiently overexpressing the human ATF4 transcription factor (two experiments), (A) or the human ATF3 transcription factor (three experiments), (B). Relative luminescence in the FNL-7 cell line expressing the human Klotho promoter driving firefly luciferase expression in control and cells overexpressing human ATF4, ATF3, or both factors (N = 6), (C). The HK-2 cells were also transduced with shRNA lentiviral vectors targeting ATF4 or ATF3 and the relative mRNA levels of Klotho as well as the protein levels were assessed by qPCR (N = 8/8/4), (D) or western blot analysis (N = 3), (E), respectively, in the presence or absence of albumin in comparison to the scrambled shRNA vector. The silencing efficiency was verified by western blot analysis (N = 4/3), (F). The mRNA levels are normalized to RPLP0 gene expression and the protein levels to β-actin or Ponceau staining. Mean ± SD, unpaired two-tailed t test or multiple t test without correction or one-way ANOVA *P < .05

Article Snippet: To isolate the lentiviral particles, the cell medium was collected 48 hours after and centrifuged at 1500 rpm for 7 minutes and passed through a Name Host and isotype Manufacturer Cat. No. Dilution Primary antibody Klotho Rat IgG2a TransGenic KO603 1:50 ATF4 Rabbit IgG Cell signaling #11815 1:250 ATF3 Rabbit IgG Abcam ab207434 1:50 Detection system Klotho ImmPRESS HRP AntiRat IgG Vector Laboratories MP-7404-50 EnVision Flex kit DAKO K8010 ATF4, ATF3 CSA II BiotinFree Catalyzed Amplification System DAKO K149711-2 T A B L E 1 Antibodies and kits used for immunohistochemistry | 5DELITSIKOU ET aL. 0.45-μM pore size filter to remove cell debris.

Techniques: Expressing, Western Blot, Luciferase, Control, Transduction, shRNA, Comparison, Plasmid Preparation, Gene Expression, Staining, Two Tailed Test

Journal: The FASEB Journal

Article Title: Klotho regulation by albuminuria is dependent on ATF3 and endoplasmic reticulum stress

doi: 10.1096/fj.201900893r

Figure Lengend Snippet: FIGURE 7 ER stress inhibition reverses Klotho downregulation. Relative mRNA levels (N = 13), (A) and western blot analysis (N = 7/8/6/9), (B) of Klotho and ER stress response genes in the cortex of control and proteinuric mice treated with vehicle or 4-PBA (1 mg/g/day in drinking water for 7 days). Representative immunohistochemical staining of Klotho, ATF4 and ATF3 in kidney sections of control and proteinuric mice, treated with 4-PBA or vehicle (C). In order to inhibit ER stress in vitro, the HEK-293 overexpressing cells were treated with 1 mM 4-PBA (N = 7) or 1 mM TUDCA (N = 5), (D) for 24 hours in the presence or absence of albumin. The Klotho protein levels were measured by western blot analysis. The mRNA levels are normalized to RPLP0 gene expression and the protein levels to Coomassie staining or β-actin. Mean ± SD, one-way or two-way ANOVA *P < .05

Article Snippet: To isolate the lentiviral particles, the cell medium was collected 48 hours after and centrifuged at 1500 rpm for 7 minutes and passed through a Name Host and isotype Manufacturer Cat. No. Dilution Primary antibody Klotho Rat IgG2a TransGenic KO603 1:50 ATF4 Rabbit IgG Cell signaling #11815 1:250 ATF3 Rabbit IgG Abcam ab207434 1:50 Detection system Klotho ImmPRESS HRP AntiRat IgG Vector Laboratories MP-7404-50 EnVision Flex kit DAKO K8010 ATF4, ATF3 CSA II BiotinFree Catalyzed Amplification System DAKO K149711-2 T A B L E 1 Antibodies and kits used for immunohistochemistry | 5DELITSIKOU ET aL. 0.45-μM pore size filter to remove cell debris.

Techniques: Inhibition, Western Blot, Control, Immunohistochemical staining, Staining, In Vitro, Gene Expression