Journal: JCI Insight

Article Title: Renal proximal tubular NEMO plays a critical role in ischemic acute kidney injury

doi: 10.1172/jci.insight.139246

Figure Lengend Snippet: ( A ) NEMO mRNA normalized to GAPDH from qRT-PCR reactions in isolated proximal tubule cells, bone marrow–derived macrophages, whole kidney, liver, spleen, and small intestine of WT NEMO fl/fl and NEMO fl/fl PEPCK-Cre mice. ( B ) NEMO protein normalized to β-actin in isolated proximal tubule cells of NEMO fl/fl and NEMO fl/fl PEPCK-Cre mice. The bands below approximately 50 kDA predicted molecular weight of NEMO may represent truncated forms, varying glycosylation products, and cleaved fragments. For statistical analysis, Student’s t test was used to detect significant changes. * P < 0.05 versus NEMO fl/fl mice ( N = 3–8). Error bars represent 1 SEM. ( C ) Confirmation of renal proximal tubule cell–specific MNP delivery. Mouse kidney sections stained with anti-PEG antibody to detect MNP localization and PHA-lectin to mark renal proximal tubule cells or with anti–aquaporin-2 antibody to mark renal collecting ducts and counterstained with DAPI to visualize cell nuclei. Mice were injected with 38 mg/kg NBP MNP or with vehicle control 6 hours before renal IR injury. Renal proximal tubular distribution pattern for PEG was confirmed by colocalization of PEG antibody staining and PHA lectin staining in MNP-injected mice (×200 original magnification images shown, representative of 3 experiments). However, PEG staining was not detected in the lung, spleen, or liver (×200 original magnification images shown, representative of 3 experiments). Scale bar: 50 μm.

Article Snippet: After blocking sections with PBS containing 10% normal rabbit serum, sections were incubated with 1:100 anti-PEG antibody (ab94764, Abcam) specific to the PEG backbone plus PHA lectin antibody (proximal tubule–specific marker, Molecular Probes) or plus aquaporin-2 antibody (collecting duct–specific marker, AQP-002, Alomone Labs).

Techniques: Quantitative RT-PCR, Isolation, Derivative Assay, Molecular Weight, Staining, Injection