pstrawberry atg4b c74a  (OriGene)

Journal: Autophagy

Article Title: Classical autophagy proteins LC3B and ATG4B facilitate melanosome movement on cytoskeletal tracks

doi: 10.1080/15548627.2017.1327509

Figure Lengend Snippet: Delipidation activity of ATG4B is crucial for translocation of melanosomes onto actin filaments at dendrites. (A) Immunofluorescence analysis for melanosome localization in B16 cells on ATG4B knockdown. Complementation with siRNA-resistant full-length ATG4B and catalytically inactive ATG4BC74A was performed. While overexpression of full-length ATG4B rescued the melanosome aggregation defect, the ATG4BC74A mutant was unable to rescue the phenotype. Scale bar: 5 μm. (B) Representative western blot for the validation of ATG4B knockdown in B16 cells. (C) Quantitation of cells with perinuclear melanosomal aggregation on siRNA-mediated silencing of ATG4B and on complementation with full length and the ATG4BC74A mutant in mouse B16 melanoma cells (no. of cells analyzed, n>100). Bars represent mean ± s.e.m. across replicates. (D) Immunofluorescence micrographs of B16 cells transfected with mCherry-LC3B∆121–125 in a siAtg4b background. Melanosomes were stained with PMEL and did not localize on actin filaments (in red) suggesting that delipidation of LC3B-II is essential for actin translocation. Scale bars: 5 μm. (E) Immunofluorescence micrographs of B16 cells transfected with mCherry-LC3B∆121–125 in the siAtg4b background. Melanosomes were stained with PMEL and were found to be present at the ends of microtubules (tubulin antibody) suggesting hampered movement to the cell periphery. Scale bars: 5 μm. (F) In vitro delipidation assay using recombinant ATG4B was performed on purified melanosomes. Conversion of LC3-II form (16 kDa) to the nonlipidated LC3-I form (18 kDa) was observed, suggesting the role of ATG4B in LC3B delipidation on melanosomes. (G) Densitometric quantification of LC3B-I and LC3B-II protein levels upon ATG4B addition to purified melanosomes shows significant differences. Reduction in LC3-II levels with parallel increase in LC3-I levels was observed (n = 4). Bars represent mean ± s.e.m. across replicates.

Article Snippet: MLANA-GFP construct was procured from Origene Technologies ( {"type":"entrez-nucleotide","attrs":{"text":"MG221327","term_id":"1316021000","term_text":"MG221327"}} MG221327 ). pStrawberry-ATG4B C74A was procured from Addgene (21076; deposited by Tamotsu Yoshimori). pGEX-6P-ATG4B was generated using EcoRI and SalI restriction sites.

Techniques: Activity Assay, Translocation Assay, Immunofluorescence, Over Expression, Mutagenesis, Western Blot, Quantitation Assay, Transfection, Staining, In Vitro, Recombinant, Purification

Journal: Autophagy

Article Title: Classical autophagy proteins LC3B and ATG4B facilitate melanosome movement on cytoskeletal tracks

doi: 10.1080/15548627.2017.1327509

Figure Lengend Snippet: LC3B localization on melanosomes shows spatial distribution in melanocytes. (A) Immunofluorescence micrographs of B16 cells immunostained with melanosome-specific monoclonal antibody HMB45 along with LC3B. Pearson correlation coefficient (PCC) and overlap coefficient (OC) between LC3B and PMEL for the entire cell were calculated (n > 10). Scale bar: 5 μm. (B) Immunofluorescence micrographs of pigmented primary human melanocyte cells immunostained with PMEL and LC3B. Melanosomes were detected under brightfield microscopy as dark granules of melanin. Overlap between each set in shown in the inset. Scale bar: 5 μm. (C) Live imaging snapshots of mCherry-LC3B coexpressed with melanosomal markers GPR143-GFP, MLANA-GFP and RAB27A-GFP showed a high degree of colocalization. The colocalization was quantified by measuring the Pearson correlation coefficient (PCC) as shown in the inset (n > 10). Scale bar: 5 μm. (D) Immunogold electron microscopy (IEM) studies confirm presence of LC3B on melanosomes. High magnification images of B16 cells immunostained with anti-LC3B antibody (secondary antibody conjugated to 10-nm gold beads) marked by white arrowheads. Scale bar: 100 nm. (E) Immunofluorescence micrograph of B16 cells immunostained with PMEL and LC3B. Pearson correlation coefficient between LC3B and PMEL at the dendrites and cell center is represented in the inset. Spatial distribution for colocalized pixels observed. Scale bar: 5 μm. N > 10. (F) Serial z-sections were imaged using confocal microscopy at approximately 500 nm from the cell periphery to cell center. Quantification of colocalized pixels is plotted as pie chart for each stack. Red represents melanosome pixels devoid of LC3B while white represents LC3B colocalized melanosomal pixels. Increased colocalization of melanosomes and LC3B-II is observed in sections spanning center of the cell. Melanosomes devoid of LC3B were mainly present at the dendritic tips. N > 25.

Article Snippet: MLANA-GFP construct was procured from Origene Technologies ( {"type":"entrez-nucleotide","attrs":{"text":"MG221327","term_id":"1316021000","term_text":"MG221327"}} MG221327 ). pStrawberry-ATG4B C74A was procured from Addgene (21076; deposited by Tamotsu Yoshimori). pGEX-6P-ATG4B was generated using EcoRI and SalI restriction sites.

Techniques: Immunofluorescence, Microscopy, Imaging, Electron Microscopy, Confocal Microscopy