Anti-Gap1 antibody. Bottom panels: Western blot with anti-Pma1 antibody as loading
Anti-Gap1 antibody. Bottom panels: Western blot with anti-Pma1 antibody as loading handle. Luminescent arbitrary units (LAU) 10-6 are shown as ratio EGFR/ErbB1/HER1 Biological Activity involving the Gap1 band and Pma1 band for every single time point to assess relative disappearance from the Gap1 band, consistent with endocytosis. The ratios among di- or tri-ubiquitinated to non-ubiquitinated Gap1 are also shown to assess the relative improve in the former with respect towards the latter right after addition of each and every nitrogen source. A Western blot from cells expressing the non-ubiquitinatable Gap1K9R,K16R subjected to identical treatment is also shown as manage to confirm that upper bands observed above the Gap1 band inside the wild-type blots are ubiquitinated forms with the transceptor.2014 The Authors. Molecular Microbiology published by John Wiley Sons Ltd., Molecular Microbiology, 93, 213222 G. Van Zeebroeck, M. Rubio-Texeira, J. Schothorst and J. M. Thevelein2014 The Authors. Molecular Microbiology published by John Wiley Sons Ltd., Molecular Microbiology, 93, 213Analogues uncouple transceptor functionsFig. 5. The non-transported and non-signalling competitive inhibitor of Gap1-mediated transport, L-Asp–L-Phe, can’t trigger endocytosis but triggers ubiquitination in the wild-type strain. A. Progressive intracellular accumulation of radioactively labelled L-Asp–L-Phe right after addition of five mM of this compound to nitrogen-starved cells. Strains: wild-type (black bars), gap1 (white bars) and opt1 dal5 ptr2 (grey bars). Error bars represent s.d. involving biological repeats. B. Development of 110 serial dilution spottings of nitrogen pre-starved cells from the strains wild-type, gap1, opt1 dal5 ptr2 and opt1 dal5 ptr2 gap1 on plates of nitrogen starvation medium (NSM) with out or supplemented with 1 mM of L-citrulline, or L-Asp–L-Phe. Exactly the same cells spotted in complete supplemented medium (CSM) are shown as good growth handle. Growth on the same cells in NSM 1 mM of your dipeptide Leu-Met-NH2 or the tripeptide DNMT1 web L-Arg-Gly-Gly is shown as control of peptide use as nitrogen source because of peptide carrier uptake. C. Localization of wild-type Gap1-GFP expressed in the strains gap1 and opt1 dal5 ptr2 gap1 is shown just before and 60, 120 and 180 min after addition of five mM L-Asp–L-Phe. The identical cells exposed to 2.5 mM L-aspartate plus two.5 mM L-phenylalanine is shown as control that the dipeptide constituent amino acids are in a position to induce endocytosis. D. Evaluation of Gap1 ubiquitination status in nitrogen-starved cells expressing endogenous GAP1 (from the wild-type or the triple deletion mutant opt1 dal5 ptr2) and induced with 10 M CuSO4 for 30 min prior to addition of nitrogen supply, for expression of myc-ubiquitin from the PCUP1-myc-Ubi URA3 plasmid, pMRT7. P13 fractions have been collected at distinctive time points (0, 30, 60, 120 and 180 min) following addition of five mM L-Asp–L-Phe to nitrogen-starved cells. Upper panels: Western blot with anti-Gap1 antibody. Bottom panels: Western blot with anti-Pma1 antibody as loading control. Luminescent arbitrary units (LAU) 10-6 are shown as ratio involving the Gap1 band and Pma1 band for each time point to assess the relative disappearance on the Gap1 band, consistent with endocytosis. The ratios in between di- or tri-ubiquitinated to non-ubiquitinated Gap1 are also shown to assess the relative increase in the former with respect towards the latter just after addition of every nitrogen supply.2002; Merhi and Andr 2012). Transport was absolutely abolished by deletion with the three major pe.