LAMP1 Antibody - #DF4806

Fig. 5| ABHD5 sustains the activity of RNASET2. a Representative images of immunofluorescent stainings of lysosome (stained by Lysotracker, red) and ABHD5 (green) in SW480 cells. Scale bar: 10 μm. b Western blots showing ABHD5 expression in the lysosome lysates of ABHD5 KD and control cells. LAMP1 was used as a reference.

Figure 1: ATP7B distribution at the TGN and endolysosomes in different copper treatments. (A) Immunofluorescence image of ATP7B (green) in HepG2 cell-co-stained with endolysosomal marker LAMP2 (red) and TGN marker TGN46 (blue) shows presence of ATP7B on both compartments, irrespective of cellular copper condition i.e., basal, high copper (100µM Cu) as well as copper chelated conditions (100µM BCS). The marked area on ‘merge’ panel is enlarged in ‘inset’. The arrowhead shows colocalization between ATP7B21 LAMP2, the asterisk shows colocalization between ATP7B-TGN46. (B) Pearson’s Colocalization Coefficient (PCC) between ATP7B-TGN46 and ATP7B-LAMP2 in different copper conditions are demonstrated by box plot with jitter points. The black * depicts the comparison of colocalization between ATP7B-TGN46 and ATP7B-LAMP2. The green * shows the comparison of ATP7B-TGN46 colocalization under different copper conditions w.r.t. basal condition. The orange * shows the comparison of ATP7B-LAMP2 colocalization under different copper conditions w.r.t. basal condition. (C) Cumulative fraction of ATP7B on TGN46 and LAMP2 positive compartment are demonstrated by box plot with jitter points. The statistical comparison is done w.r.t. Basal. Sample size (N) for (B) and (C) are TTM: 42, BCS: 57, Basal: 24, Cu20: 44, Cu100: 49, Cu250: 50. (D) comparison of copper accumulation in HepG2 cells (n = 9) under different treatment conditions shows elevated copper accumulation in case of increased copper treatment, copper concentrations were measured in parts per billion (ppb). Data is demonstrated as bar-plot (mean ± SD). Statistical comparison is done w.r.t basal. (E) Comparison of copper accumulation in HepG2 cells (n = 9) under prolong copper chelated conditions (12h as well as 72h chronic chelation using BCS) shows decreased copper accumulation. Copper concentrations were measured in parts per billion (ppb). Data is demonstrated as bar-plot (mean ± SD). (F) Immunofluorescence image of ATP7B (green) in HepG2 cell line co-stained with endolysosomal marker LAMP2 (red) and TGN marker TGN46 (blue) shows presence of ATP7B on both the compartments even under chronic copper chelated condition. The marked area on ‘merge’ panel is enlarged in ‘inset’. The arrowhead shows colocalization between ATP7B-LAMP2, the asterisk shows colocalization between ATP7B-TGN46. (G) Immunofluorescenc image of ATP7B (green) co-stained with LAMP2 (red) in mouse tissue shows presence of ATP7B on LAMP2 positive compartment (marked by arrowhead). (H) Immuno-EM of endogenous ATP7B in HepG2 cell show presence of ATP7B at the Golgi membrane (marked by arrow) as well as late-endosome/lysosome compartmen (marked by arrowhead) both in BCS treated (left panel) and basal (right panel) conditions. (I) Immunoblot of ATP7B, lysosomal marker LAMP2, TGN marker GGA2 and TGN46, early endosome marker EEA1 and late endosome marker Rab7 of the top 8 fractions of sucrose gradient shows presence of ATP7B on TGN and lysosome enriched fraction. The line plot shows abundance of ATP7B (green), GGA2 (orange) and LAMP2 (violet) in 8 fractions of sucrose gradient. The ribbon plot signifies the standard deviation of 4 independent experiments. [scale bar for EM: 100 nm, for immunofluorescence: 5µm]