Journal: American Journal of Physiology - Cell Physiology
Article Title: Knockout of the neonatal Fc receptor in cultured podocytes alters IL-6 signaling and the actin cytoskeleton
doi: 10.1152/ajpcell.00235.2019
Figure Lengend Snippet: There is no difference in RhoA, inverted formin 2 (INF2), and kidney ankyrin repeat-containing protein 2 (KANK2) expression in wild type (WT) or knockout (KO) podocytes at baseline or after treatment with proinflammatory stimuli. A–D: Western blot analysis of RhoA in WT and neonatal Fc receptor (FcRn) KO podocytes after treatment with regular media (RM), IFNɣ, 4-hydroxy-3-nitrophenylacetyl hapten-ovalbumin (NP-ova), or IFNɣ plus NP-ova for 30 min (A and B, n = 3 experiments) and 48 h (C and D, n = 2 experiments), respectively. Data are presented as means ± SE. E and F: Western blotting of INF2 expression after treatment with regular media (RM), IFNɣ, NP-ova, or IFNɣ plus NP-ova for 30 min (n = 4 experiments). Data are presented as means ± SE. G and H: Western blotting of KANK2 expression after treatment with regular media (RM), IFNɣ, NP-ova or IFNɣ plus NP-ova for 30 min (n = 3 experiments). Data are presented as means ± SE. In B, D, F, and H, ns, not statistically significant. I: RhoA activity in WT and FcRn KO podocytes after treatment with regular media, IFNɣ, NP-ova, or NP-ova + IFNɣ for 30 min.
Article Snippet: Membranes were blocked (5% of nonfat milk, 60 min) and were then incubated overnight with a 1:1,000 dilution of primary antibodies including: rabbit anti-mouse STAT3 antibody (cat. no. 12640s, Cell Signaling Technology), rabbit anti-mouse phospho-STAT3 (Y705) (cat. no. 9145s, Cell Signaling Technology), rabbit anti-mouse STAT1 antibody (cat. no. 14994S, Cell Signaling Technology), rabbit anti-mouse phospho-STAT 1 antibody (cat. no. 9167S, Cell Signaling Technology), rabbit anti-mouse synaptopodin antibody (cat. no. 21064-1-AP, Proteintech), rabbit anti-mouse Ras homolog gene family, member A (RhoA) antibody (cat. no. 2117s, Cell signaling Technology) rabbit anti-mouse kidney ankyrin repeat-containing protein 2 (KANK2) antibody (cat. no. 21733-1-AP, Proteintech), and rabbit anti-mouse inverted formin 2 (INF2) antibody (cat. no. 20466-1-AP, Proteintech), at 4°C.
Techniques: Expressing, Knock-Out, Western Blot, Activity Assay
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