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

Journal: International Journal of Nanomedicine

Article Title: Precision USPIO-PEG-SLe x Nanotheranostic Agent Targeted Photothermal Therapy for Enhanced Anti-PD-L1 Immunotherapy to Treat Immunotherapy Resistance

doi: 10.2147/IJN.S445879

Figure Lengend Snippet: The images of immunohistochemical (IHC) staining assessing Ki67 expression and immunofluorescence analysis of CRT and HMGB1 about the primary tumor tissues. ( A ) IHC staining assessing Ki67 expression of the tumor cells proliferation. Scale bars represent 100 μm. ( B ) Immunofluorescence analysis of calreticulin (CRT) expression from primary tumor tissues of the four different groups (scale bar: 10 μm). ( C ) Fluorescence quantitative fluorescence analysis of CRT. ( D ) Immunofluorescence analysis of high mobility group box-1 protein (HMGB1) expression from primary tumor tissues of the four different groups (scale bar: 10 μm). ( E ) Quantitative fluorescence analysis of HMGB1. Three randomly chosen visual fields were evaluated for histological quantification. Data are represented as mean ± SD.

Article Snippet: 3’-sialyl Lewis X (termed SLe x ) (Carbosynth, UK), Fe 3 O 4 @OA@DSPE-PEG2k-NH 2 (Nanjing Nanoeast Biotech Co., Ltd.), 2- (N-morpholino) ethanesulfonic acid (MES) (TMLT40545500MG, China National Pharmaceutical Group Corporation Chemical Reagent Company), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) (Sigma), 808 nm near-infrared laser (BWT, Beijing, China), anti-mouse PD-L1 (B7-H1) in vivo monoclonal antibody (Selleck), high mobility group box 1 (HMGB1) antibody (AF7020, Affinity Biosciences Ltd.), calreticulin (CRT) antibody (DF6211, Affinity Biosciences Ltd.), recombinant anti-CD4 antibody (ab183685, Abcam), recombinant anti-CD8 alpha antibody (ab217344, Abcam).

Techniques: Immunohistochemical staining, Immunohistochemistry, Expressing, Immunofluorescence, Fluorescence