Journal: Nature Communications
Article Title: Caveolin-1 mediates cellular distribution of HER2 and affects trastuzumab binding and therapeutic efficacy
Figure Lengend Snippet: Seesaw effect between CAV1 and HER2 proteins. a Association between CAV1 and HER2 in gastric (NCIN87, KATOIII), bladder (HT1197, UMUC14, UMUC3), breast (MDAMB231, MCF-7, BT474, SKBR3), pancreatic (BxPc3, MiaPaCa, Suit2) and prostate (LnCap, Pc3) cancer cells. The level of CAV1 ( y -axis) and HER2 ( x -axis) are expressed as the ratio of protein/β-actin control, as determined by western blot analysis (upper panel). The non-parametric one-tailed Spearman test was used to determine the correlation coefficients. R HER2/CAV1 , ratio of HER2-to-CAV1 protein levels as determined by western blot analysis from total extracts (lower panel). b Confocal images of immunofluorescence staining of HER2 in s.c. NCIN87 gastric tumors from athymic nude mice, showing that HER2 does not exhibit a predominant membrane staining. Scale bars: 100 μm and 50 μm (inset). c Confocal images of immunofluorescence staining of HER2 and CAV1 in s.c. NCIN87 gastric tumors from athymic nude mice. HER2 presence at the cell membrane in cells where CAV1 is absent (cell clusters 1 and 3) and HER2 almost absent at the cell membrane in cells where CAV1 is present (cell clusters 2 and 4). Scale bars: 50 μm. d Confocal images and quantification (mean ± S.E.M, n = 3) of immunofluorescence staining of HER2 and CAV1 in human HER2-positive gastric tumors containing high (Patient 1) and low (Patient 2) expression of CAV1. HER2 predominant presence at the cell membrane in CAV1-negative tumors (Patient 2). Scale bars: 50 μm. e Western blot analysis of the distribution of HER2 and CAV1 in caveolae fractions isolated from NCIN87 cells. f Western blot of HER2 and CAV1 in the total lysates of NCIN87 cells after blocking protein synthesis with 80 μg mL −1 CHX for 0, 12, 24, 30, 48, 60, 72, and 96 h. CHX cyclohexamide. g HER2 and CAV1 have similar half-lives. Half-lives calculated after western blot analysis of Fig. 1f. Density of western blot bands was quantified by scanning densitometry with ImageJ software. Half-lives were calculated as the time required for protein decrease to 50% of its initial level (mean ± S.E.M, n = 4)
Article Snippet: Following electrophoresis and transfer to polyvinylidene difluoride (PVDF) membranes (Bio-Rad), the blots were incubated in 5% (m/v) BSA in Tris-buffered saline buffer-Tween (TBS-T, Cell Signaling Technology) and probed with mouse anti β-actin 1:20,000 (Sigma, A1978), rabbit anti-HER2 1:800 (Abcam, ab131490), rabbit anti-H ER2 phospho Y1139 1:500 (Abcam, ab53290), and rabbit anti-CAV1 1:500 (Abcam, ab2910) antibodies.
Techniques: Western Blot, One-tailed Test, Immunofluorescence, Staining, Mouse Assay, Expressing, Isolation, Blocking Assay, Software