Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: WD40-repeat 47, a microtubule-associated protein, is essential for brain development and autophagy

doi: 10.1073/pnas.1713625114

Figure Lengend Snippet: SCG10 interacts with WDR47. (A) Colocalization images of WDR47 with SCG10 and SCG10 with JNK1. (B) Relative expression of SCG10 transcripts in n = 3 Wdr47tm1a/tm1a mice using qRT-PCR compared with WT across four tissues (cortex, spinal cord, thalamus, and liver) plotted as mean + SEM. (C) Colocalization of SCG10 and WDR47 in GT1-7 hypothalamic neuronal cells. Upper Left shows expression in GT1-7 cells transfected with pEYFP-WDR47 (green). Upper Right shows expression of SCG10 labeled with Texas red anti-SCG10 antibody (red). Lower Left is the overlay of Upper, and Lower Right is the 3D colocalization of WDR47 and SCG10. (D) Western blot analysis of SCG10 relative protein levels in response to WDR47 siRNA treatment. GAPDH is used as a loading control. Statistical analysis was done using Student’s t test (two-tailed). *P < 0.05. (Magnification: A, Top and Bottom, 20×/one dry objective; A, Middle, 65×/one oil objective.)

Article Snippet: The cells were stained using a range of antibodies: rabbit polyclonal anti-WDR47 (ab121935; 1:50 dilution; Abcam); rabbit polyclonal anti-MAP2 (AB5622; 1:1,000 dilution; Millipore), which stains the cell body and dendrites; mouse monoclonal antineurofilament panaxonal antibody (SMI-312R; Covance and 837904; 1:1,000 dilution; Biolegend), which stains the axon and growth cone; mouse monoclonal anti-STMN2 (MABN663; 1:50 dilution; Merck Millipore); goat polyclonal anti-JNK1 (sc-46006; 1:50 dilution; Santa Cruz Biotechnology); and mouse monoclonal antiacetylated tubulin clone 6–11b-1 (T6793; 1:200 dilution; Sigma-Aldrich), a marker for stabilized microtubules.

Techniques: Expressing, Quantitative RT-PCR, Transfection, Labeling, Western Blot, Two Tailed Test

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: WD40-repeat 47, a microtubule-associated protein, is essential for brain development and autophagy

doi: 10.1073/pnas.1713625114

Figure Lengend Snippet: Summary of WDR47 working model. At the cellular level, WDR47 acts as a stabilizer of microtubules. SCG10, a microtubule destabilizer at the growth cone, is regulated by JNK1 through phosphorylation. In the cytoplasm, WDR47 negatively regulates p62 and LC3-mediated autophagy. Reduced expression of Wdr47 leads to destabilization of the microtubules, increased Tau and autophagic flux, and decreased cell motility. At the tissue level, the loss of Wdr47 yields fiber tract defects, including corpus callosum agenesis, microcephaly, and thinner cortices, that are linked to a reduced number of progenitors, increased number of neurons in the intermediate zone, and elevated number of dying neurons in the cortical upper layers. At the organism level, these defects may explain impairment in motor coordination, reduced grip strength, and increased pain sensitivity. AP, apical progenitor; IP, intermediate progenitor; MN, migrating neuron; MT, microtubule.

Article Snippet: The cells were stained using a range of antibodies: rabbit polyclonal anti-WDR47 (ab121935; 1:50 dilution; Abcam); rabbit polyclonal anti-MAP2 (AB5622; 1:1,000 dilution; Millipore), which stains the cell body and dendrites; mouse monoclonal antineurofilament panaxonal antibody (SMI-312R; Covance and 837904; 1:1,000 dilution; Biolegend), which stains the axon and growth cone; mouse monoclonal anti-STMN2 (MABN663; 1:50 dilution; Merck Millipore); goat polyclonal anti-JNK1 (sc-46006; 1:50 dilution; Santa Cruz Biotechnology); and mouse monoclonal antiacetylated tubulin clone 6–11b-1 (T6793; 1:200 dilution; Sigma-Aldrich), a marker for stabilized microtubules.

Techniques: Expressing