Journal: PLoS ONE

Article Title: A Knockout of the Tsg101 Gene Leads to Decreased Expression of ErbB Receptor Tyrosine Kinases and Induction of Autophagy Prior to Cell Death

doi: 10.1371/journal.pone.0034308

Figure Lengend Snippet: A . Quantitative real-time RT-PCR to assess the transcriptional activation of the Microtubule-associated protein 1/light chain 3 ( Map1lc3 ) gene in Tsg101 conditional knockout cells and their wildtype controls. B . Western blot analysis to verify the increase in expression of LC3 in response to Tsg101 deficiency. Wildtype cells maintained in the absence of growth factors (GF) were used as a positive control. C ., D . Analysis of confocal images of Tsg101 conditional knockout cells and their wildtype controls that were maintained in the presence or absence of growth factors (GF) prior to fixation and co-staining with antibodies against endogenous LC3 and Lamp1. Panel C illustrates the quantitative analysis of the colocalization of LC3 and Lamp1 ( P value, t test). The bar in panel D represents 10 µm and arrows indicate areas of intensive co-localization of LC3 and Lamp1.

Article Snippet: The following antibodies were used in this study: α-Tsg101 (C-2) (1∶1000 dilution), α-ActB (I-19)(1∶1000 dilution) and α-EGFR p-Tyr1173 (sc-12351) from Santa Cruz Biotechnology; α-EGFR (1∶200 dilution) from AbCam; α-ErbB2 (Her2/Neu) (OP-15) (1∶1000 dilution) from Calbiochem; α-phospho-ERK (1∶5000 dilution) from Cell Signaling Technology, α-pan-ERK (1∶2000 dilution) from BD Bioscience; and α-Map1LC3 (4E12) (1∶2000 dilution) from MBL International.

Techniques: Quantitative RT-PCR, Activation Assay, Knock-Out, Western Blot, Expressing, Positive Control, Staining