Functional analyses of <i>MAN1B1</i> mutations. Daisy Rymen Romain Peanne María B. Millón Valérie Race Luisa Sturiale Domenico Garozzo Philippa Mills Peter Clayton Carla G. Asteggiano Dulce Quelhas Ali Cansu Esmeralda Martins Marie-Cécile Nassogne Miguel Gonçalves-Rocha Haluk Topaloglu Jaak Jaeken François Foulquier Gert Matthijs 10.1371/journal.pgen.1003989.g005 https://plos.figshare.com/articles/figure/_Functional_analyses_of_MAN1B1_mutations_/876440 <p>(<b>A</b>) Quantification of the <i>MAN1B1</i> transcript in controls and affected individuals by qPCR without (left panel) and with (right panel) puromycin. <i>MAN1B1</i> expression was normalized to the expression of the house-keeping gene <i>HPRT</i>. Values plotted with a wild-type control untreated with puromycin are set to 1. The depicted values are the mean ± SEM of at least three independent experiments. (<b>B</b>) Steady-state levels of the expression of MAN1B1 in control (C) and MAN1B1-deficient (P) fibroblasts. Whole-cell extracts were analysed by immunoblotting using anti-MAN1B1 antibodies. Thirty micrograms of total cell extracts were loaded into each lane and anti-β-actin antibodies were used as a loading control. Quantifications represent the mean value of three independent loadings of three independent samples. (<b>C</b>) Intracellular distribution of MAN1B1 in control and MAN1B1-deficient fibroblasts. Fibroblasts were double-labelled with antibodies against MAN1B1 (green) and the Golgi marker giantin (red), then fixed and processed for analysis by confocal laser scanning microscopy. Images were collected under identical settings. Depicted are the zoomed images. The independent panels are presented in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003989#pgen.1003989.s004" target="_blank">Figure S4</a>.</p> 2013-12-12 03:59:46