Journal: American Journal of Physiology - Renal Physiology

Article Title: Regulation of the creatine transporter by AMP-activated protein kinase in kidney epithelial cells

doi: 10.1152/ajprenal.00162.2010

Figure Lengend Snippet: Creatine transporter (CRT) and AMP-activated protein kinase (AMPK) localize at the apical pole in rat kidney proximal tubules. A–C: confocal microscopic images of control rat kidney immunolabeled using an anti-CRT antibody alone (A, green) or an anti-CRT antibody amplified by tyramide signal amplification (B) and then using an anti-AMPK-α antibody (C, red). D: regions of colocalization of CRT and AMPK-α at the apical pole of proximal tubule cells (yellow). Scale bars, 100 μm. E–G: little nonspecific staining in tissues incubated in the absence of primary antibody.

Article Snippet: Custom-made immunopurified anti-CRT antibodies recognizing the NH 2 terminus of the CRT were obtained by immunization of rabbits with a synthetic peptide comprising amino acids 14–27 of rat SLC6A8 (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"NM_017348.2","term_id":"132626536","term_text":"NM_017348.2"}} NM_017348.2 , Anawa, Switzerland).

Techniques: Control, Immunolabeling, Amplification, Staining, Incubation

Journal: American Journal of Physiology - Renal Physiology

Article Title: Regulation of the creatine transporter by AMP-activated protein kinase in kidney epithelial cells

doi: 10.1152/ajprenal.00162.2010

Figure Lengend Snippet: AMPK activation inhibits apical pole localization of CRT in mouse S3 proximal tubule cells, as determined by immunofluorescence confocal microscopy. A: time course of 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR)-mediated AMPK activation, as measured by immunoblotting of phosphorylated (Thr172) AMPK-α (pT172) in S3 cell lysates. AICAR treatment (1 mM) for 2–16 h induced a significant upregulation of phosphorylated (Thr172) AMPK-α compared with untreated cells at time 0 under conditions of equivalent protein loading (β-actin control). B: AICAR-induced cytoplasmic redistribution of CRT in polarized S3 cells grown on Transwell filters, as shown by x-z reconstructions of confocal microscope image stacks of S3 cell monolayers immunolabeled using anti-CRT antibody (green) alone or anti-CRT antibody and an anti-ZO-1 antibody (red) grown under control conditions (Con) or in the presence of 1 mM AICAR for 2 h. C: Cy3-coupled phalloidin staining of S3 cell monolayers treated with vehicle (Con) or 1 mM AICAR for 2 h. Images are representative of ≥3 different filter sets and treatments. Scale bar, 10 μm.

Article Snippet: Custom-made immunopurified anti-CRT antibodies recognizing the NH 2 terminus of the CRT were obtained by immunization of rabbits with a synthetic peptide comprising amino acids 14–27 of rat SLC6A8 (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"NM_017348.2","term_id":"132626536","term_text":"NM_017348.2"}} NM_017348.2 , Anawa, Switzerland).

Techniques: Activation Assay, Immunofluorescence, Confocal Microscopy, Western Blot, Control, Microscopy, Immunolabeling, Staining

Journal: American Journal of Physiology - Renal Physiology

Article Title: Regulation of the creatine transporter by AMP-activated protein kinase in kidney epithelial cells

doi: 10.1152/ajprenal.00162.2010

Figure Lengend Snippet: Complex signal pattern of CRT revealed by immunoblotting. HEK-293 (lanes 1–4) or S3 (lanes 5–6) cells were transiently transfected with empty vector (Con) or a plasmid encoding the hemagglutinin (HA)-tagged CRT. One-day-posttransfection cell lysates were separated by SDS-PAGE and analyzed by immunoblotting using the anti-CRT (lanes 1 and 2) or anti-HA (lanes 3–6) antibody. Both antibodies revealed similar immunoblot signal patterns after transfection with HA-CRT in HEK-293 cells, identifying 4 major bands (short arrows on left). However, the signal pattern observed in HA-CRT-transfected S3 cells (lane 6; long arrows on right) differed from that in HA-CRT-transfected HEK-293 cells (lane 4), suggesting that cellular processing of the CRT at the mRNA and/or protein level differs as a function of host cell type.

Article Snippet: Custom-made immunopurified anti-CRT antibodies recognizing the NH 2 terminus of the CRT were obtained by immunization of rabbits with a synthetic peptide comprising amino acids 14–27 of rat SLC6A8 (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"NM_017348.2","term_id":"132626536","term_text":"NM_017348.2"}} NM_017348.2 , Anawa, Switzerland).

Techniques: Western Blot, Transfection, Plasmid Preparation, SDS Page