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 control. Luminescent arbitrary units (LAU) 10-6 are shown as ratio involving the Gap1 band and Pma1 band for every single time point to assess relative disappearance from the Gap1 band, constant with endocytosis. The ratios involving di- or tri-ubiquitinated to non-ubiquitinated Gap1 are also shown to assess the relative increase in the former with respect for the latter after addition of every single nitrogen supply. 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 Bim MedChemExpress wild-type blots are ubiquitinated types of your transceptor.2014 The Authors. Molecular Cereblon list 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. five. The non-transported and non-signalling competitive inhibitor of Gap1-mediated transport, L-Asp–L-Phe, can not trigger endocytosis but triggers ubiquitination in the wild-type strain. A. Progressive intracellular accumulation of radioactively labelled L-Asp–L-Phe immediately 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. Growth of 110 serial dilution spottings of nitrogen pre-starved cells of the strains wild-type, gap1, opt1 dal5 ptr2 and opt1 dal5 ptr2 gap1 on plates of nitrogen starvation medium (NSM) with no or supplemented with 1 mM of L-citrulline, or L-Asp–L-Phe. The same cells spotted in total supplemented medium (CSM) are shown as good growth manage. Development of the identical cells in NSM 1 mM from the dipeptide Leu-Met-NH2 or the tripeptide L-Arg-Gly-Gly is shown as manage of peptide use as nitrogen supply resulting from peptide carrier uptake. C. Localization of wild-type Gap1-GFP expressed within the strains gap1 and opt1 dal5 ptr2 gap1 is shown prior to and 60, 120 and 180 min after addition of 5 mM L-Asp–L-Phe. The identical cells exposed to two.5 mM L-aspartate plus 2.5 mM L-phenylalanine is shown as control that the dipeptide constituent amino acids are able to induce endocytosis. D. Analysis of Gap1 ubiquitination status in nitrogen-starved cells expressing endogenous GAP1 (in the wild-type or the triple deletion mutant opt1 dal5 ptr2) and induced with ten M CuSO4 for 30 min prior to addition of nitrogen supply, for expression of myc-ubiquitin in the PCUP1-myc-Ubi URA3 plasmid, pMRT7. P13 fractions have been collected at various time points (0, 30, 60, 120 and 180 min) following addition of 5 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 amongst the Gap1 band and Pma1 band for every single time point to assess the relative disappearance with the Gap1 band, consistent with endocytosis. The ratios between di- or tri-ubiquitinated to non-ubiquitinated Gap1 are also shown to assess the relative increase on the former with respect to the latter following addition of every nitrogen source.2002; Merhi and Andr 2012). Transport was completely abolished by deletion in the 3 big pe.