Journal: The Journal of Biological Chemistry
Article Title: Stat3-mediated alterations in lysosomal membrane protein composition
Figure Lengend Snippet: Stat3-mediated regulation of the lysosomal compartment. A , validation by Western blot analysis of the lysosomal localization of flotillin 1 and flotillin 2 using iron nanoparticles to isolate EpH4 lysosomes (Mag+) after 72 h of OSM stimulation. White asterisk , indicates the expected molecular weight of flotillin 2. Representative blots of n = 3 (flotillin 1) and n = 2 (flotillin 2) independent experiments are shown. B , microarray analysis of 12 different timepoints during the mammary gland pregnancy cycle showing the involution-related expression profiles of flotillin 1 and flotillin 2. V , virgin; d G , days gestation; d L , days lactation; h I , hours involution. C , fluorescence microscopy of LAMP1 and LAMP2 immunostaining in EpH4 cells treated with OSM for 72 h. Scale bars : 10 μm. D , Western blot analysis of OSM-induced changes to the lysosomal proteins LAMP1, LAMP2, CD63, and cathepsin B in whole-cell lysates from normal (WT) and two independent Stat3 KO EpH4 cell lines after 72 h of stimulation. Proteins were separated by SDS-PAGE under reducing or non-reducing conditions as indicated. pro-Ctsb , pro form of cathepsin B; sc Ctsb , single-chain cathepsin B; Scram , scrambled sgRNA control EpH4 cells. E , microarray analysis of 12 different timepoints during the mammary gland pregnancy cycle showing the involution-related expression profiles of CD63 and LAMP2.
Article Snippet: The following primary antibodies were used: rat anti-LAMP2 (ab13524, Abcam, 1:1,000), goat anti-cathepsin L (AF1515, R & D Systems, 1:1,000), goat anti-cathepsin B (AF965, R & D Systems, 1:1,000), mouse anti-COX IV (ab33985, Abcam, 1:1,000), rabbit anti-phosphatidylinositol 3-kinase, p85 (06-496, Millipore, 1:1000), rabbit anti-Stat3 (sc-482, Santa Cruz Biotechnology, 1:1000), rabbit anti-phospho-Stat3 (9131, Cell Signaling, 1:1000), mouse anti-ERp57 (sc-23886, Santa Cruz Biotechnology, 1:400), rabbit anti-GAPDH (5174, Cell Signaling, 1:1000), goat anti-histone H3 (sc-8654, Santa Cruz Biotechnology, 1:1000), rabbit anti-Rab5 (2143, Cell Signaling, 1:1000), rat anti-LAMP1 (ID4B, Developmental Studies Hybridoma Bank, 1:200), rat anti-CD63 (NVG-2, 143901, Biolegend, 1:1000), rabbit anti-flotillin 1 (18634, Cell Signaling Technology, 1:800), and rabbit anti-flotillin 2 (HPA0013961, Atlas Antibodies, 1:200).
Techniques: Western Blot, Molecular Weight, Microarray, Expressing, Fluorescence, Microscopy, Immunostaining, SDS Page