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Fig. 1 | Molecular Neurodegeneration

Fig. 1

From: Network approach identifies Pacer as an autophagy protein involved in ALS pathogenesis

Fig. 1

Pacer is identified as a protein involved in autophagy in the context of an ALS disease network. a Scheme for the convergent analysis performed for ALS data. CNV, copy number variation; IPA, Ingenuity pathway analysis. b Scheme of localization of a total of 8 peptides (dark gray boxes) used to generate an antibody specific for mouse Pacer. Only one peptide (pink box) resulted in the generation of a specific antibody. The aa sequence of this peptide is shown. c, The specificity of the antibody generated in b was tested by depleting NSC34 cells of Pacer expression using two shRNA constructs targeting mouse Pacer mRNA (shPacer A and shPacer B). As a mock control, a scrambled shRNA (shCtrl) construct was used. Pacer was detected by a custom-made antibody by Western blot, a non-transfected (NT) is shown. A representative of 3 independent experiments is shown. d, e NSC34 cells were treated with rapamycin for 6 h. d mRNA extraction and quantitative PCR was performed (n = 4). e, Extracts of NSC34 cells were subjected to Western blot analysis. Protein levels of Pacer, Beclin1, and LC3-II were verified. β-Actin serves as a loading control. f IP was performed with extracts from HEK293T cells transfected with expression vector for mouse Pacer-V5 or empty control vector for 48 h. IP was performed using the V5 tag. The interaction of V5-tagged Pacer with endogenous Beclin1 was analyzed by Western blot. The inputs and elutions are shown. g, h The colocalization of endogenous Pacer and Beclin1 in NSC34 cells under basal (NT) and Rapamycin (Rapa) treatment was was analyzed by g, confocal microscopy and h, quantified using Pearson’s coefficient (n = 3). The nuclei are visualized with Hoechst. Mean and SEM with only statistical significant p-values are shown: *, p ≤ 0.05

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