Journal: Cellular Logistics

Article Title: Interaction with the effector dynamin-related protein 1 (Drp1) is an ancient function of Rab32 subfamily proteins

doi: 10.4161/21592799.2014.986399

Figure Lengend Snippet: Localization of Rab32 Family Proteins to the ER, Mitochondria and Lysosomes. A-C. HeLa cells were processed for confocal immunofluorescence microscopy for Rab32 family proteins and the ER (detected with ERp57, ( A ), mitochondria (visualized with Mitotracker, ( B ) and lysosomes (stained with LAMP-1, ( C ). Yellow color in the overlap indicates apposition of the Rab and ER or mitochondria staining. Pictures correspond to extended focus images. Scale bar = 25 μm. Representative images are shown. ( D). Co-localization was quantified using the Manders coefficient for the localization of Rabs with the ER, mitochondria and lysosomes, as shown by representative images in panels A-C. P a = 0.0011, P b = 0.0064, P c = 0.0001, P d = 0.0004. (Standard deviation is shown, n = 6) (E). Rab32 family distribution between microsomes (containing ER, endosomes and lysosomes), mitochondria and the MAM. HeLa homogenates were fractionated via the Percoll fractionation protocol into cytosol, microsomes, pure mitochondria, and MAM, as indicated on the right side. Loading was as follows: Homogenate 0.6% of total, cytosol, microsomes, pure mitochondria, MAM (all 2.4% of total).

Article Snippet: They have been purchased as follows: rabbit anti-Rab29, rabbit anti-Rab32 (Sigma Prestige Antibodies, St. Louis, MO), rabbit anti-Rab38 (abcam, Cambridge UK ab155956, used for IF), mouse anti-Rab38 (Abnova, Taipei, Taiwan, used for Western blot), rabbit anti-PKA RII, rabbit anti-LAMP1 (Santa Cruz Biotechnology, Dallas, TX), mouse anti-ERp57 (StressMarq, Victoria, BC), rabbit anti-Drp1 (Cell Signaling, Danvers, MA), rabbit anti-Syntaxin 7 (ProteinTech, Chicago, IL), mouse and rabbit anti-FLAG (Rockland, Gilbertsville, PA; Sigma, Oakville, ON), goat anti-FACL4, rabbit anti-Rab7, mouse anti-complex 2 (abcam, Cambridge, UK), mouse anti-PDI, (Thermo-Pierce, Rockford, IL), rabbit anti-calnexin antibody (our lab ).

Techniques: Immunofluorescence, Microscopy, Staining, Standard Deviation, Fractionation

Journal: Endocrinology

Article Title: Deletion of glycogen synthase kinase-3β in cartilage results in up-regulation of glycogen synthase kinase-3α protein expression.

doi: 10.1210/en.2010-1412

Figure Lengend Snippet: FIG. 6. GSK-3 regulates p57 expression in the prehypertrophic zone in vitro, whereas cartilage-specific GSK-3 deletion does not affect p57 expression in vivo. A, IHC analyses of p57 expression in paraffin sections of tibia organ culture treated with GSK-3 inhibitor (10 M SB86), PI3K inhibitor (10 M LY), a combination of both inhibitors (10 M SB86 plus 10 M LY), or DMSO [control (Cont)]. Black boxed insets are higher-magnification images depicting the zones of p57 staining (black arrows). Far right image is a higher-magnification (boxed area) of staining to determine cellular localization. B, Quantification of the length (micrometers) of the zone of p57 staining observed in A. Data were analyzed by one-way ANOVA with a Tukey posttest with significance denoted by asterisks (n 3; *, P 0.01). C, IHC analysis of p57 expression in paraffin sections of P0 tibias of cartilage-specific GSK-3 KO mice compared with littermate controls (Cont).

Article Snippet: Materials and Methods Materials The following antibodies were used in this study: actin A5441 (Sigma Chemical Co., St. Louis, MO); cyclin D1 RM-9104-S1 (Neomarkers, Fremont, CA); goat antirabbit horseradish peroxidase (hrp) sc-2004, goat antimouse hrp sc-2005, p57/Kipp2 sc-8298 (Santa Cruz Biotechnology, Santa Cruz, CA); GSK-3 no. 9315, pGSK-3 no. 9336, GSK-3 no. 9338, pGSK-3 and - no. 9331, and -catenin no. 9562 (Cell Signaling Technology, Danvers, MA).

Techniques: Expressing, In Vitro, In Vivo, Organ Culture, Control, Staining