Journal: Autophagy

Article Title: WIPI1 promotes fission of endosomal transport carriers and formation of autophagosomes through distinct mechanisms

doi: 10.1080/15548627.2021.1886830

Figure Lengend Snippet: Antibodies used

Article Snippet: LAMP1 , H4A3 , H4A3 , USBiological Life Sciences , 1/500; 1/1000 , IF WB.

Techniques: Transduction

Journal: PLoS ONE

Article Title: The Pharmacological Chaperone AT2220 Increases the Specific Activity and Lysosomal Delivery of Mutant Acid Alpha-Glucosidase, and Promotes Glycogen Reduction in a Transgenic Mouse Model of Pompe Disease

doi: 10.1371/journal.pone.0102092

Figure Lengend Snippet: ( A ) GAA and glycogen levels were assessed in tissue lysates prepared from heart and gastrocnemius of 12-week old male Gaa KO, hP545L GAA Tg/KO, and wild-type (denoted as ‘WT’ in the figure) mice. Significant differences in GAA activity and glycogen levels in hP545L GAA Tg/KO and Gaa KO mice compared to wild-type mice are reported as *p<0.05, t-test. Significant differences in GAA activity and glycogen levels in hP545L GAA Tg/KO mice compared to Gaa KO mice are reported as # p<0.05, t-test. Each bar represents the mean ± SEM from four mice per group analyzed in triplicate. Glycogen accumulation ( B ) and lysosomal proliferation (assessed by the quantity of LAMP1) ( C ) were assessed histochemically in Gaa KO, hP545L GAA Tg/KO, and wild-type (denoted as ‘WT’ in the figure) mouse cardiomyocytes and skeletal muscles fibers of the gastrocnemius. Glycogen staining is represented as dark pink spots, and LAMP1 staining as dark brown spots (each denoted with black arrows). The data shown are representative photomicrographs from four mice using a 40× (scale bar: 50 µm) or 20× (scale bar: 100 µm) objective in ( B ) and ( C ), respectively.

Article Snippet: After an overnight incubation with a rat anti-mouse LAMP1 antibody (1∶500 dilution) at 4°C, sections were incubated with components from the Promark rat-on-mouse HRP-polymer kit (Biocare Medical); stain was developed with a Betazoid DAB kit (Biocare Medical).

Techniques: Activity Assay, Muscles, Staining

Journal: The Journal of Cell Biology

Article Title: The late endosomal p14–MP1 (LAMTOR2/3) complex regulates focal adhesion dynamics during cell migration

doi: 10.1083/jcb.201310043

Figure Lengend Snippet: Arl8b-dependent MT plus end–directed transport of late endosomes regulates FAs. (A) MP1 endosomes are transported along MTs. Time-lapse images of HeLa cells expressing GFP-MP1 (green), mCherry-Paxillin (red), and mCherry-tubulin (red) show colocalization of MP1 and MTs (white arrowheads). Representative individual endosome moves along MTs toward two FAs (bottom panels, white arrowheads). See also Video 7 and 8 . (B) Nocodazole treatment of a cell transfected as in A results in MT depolymerization and “trapping” of few GFP-MP1 endosomes in FAs (white arrows and arrowheads). Time-lapse images of the same cell show that positions of GFP-MP1 endosomes do not change in time due to abolished MP1 transport. (C) Arl8b knockdown in MEFs. IF: anti-LAMP1 (green), anti-tubulin (red) antibodies, and Hoechst. LAMP1-positive late endosomes collapse to the perinuclear region upon Arl8b knockdown (white arrow). WB: anti-Arl8b antibody, anti-tubulin used as loading control. (D) The p14 −/− ;p14-GFP MEFs treated with control and Arl8b RNAi. The late p14-GFP endosomes cluster in the Arl8b RNAi-treated cells (red arrow). See also Video 9 . (E) The graph on the left shows the quantification of average FA length in MEFs. Mean in percent ± SEM compared with control p14 f/− MEFs treated with control RNAi (mean of FA length in control p14 f/− MEFs treated with control RNAi was taken as 100%). See also Table S1 . The graph on the right shows the migration speed of p14 −/− ;p14-GFP MEFs transfected with control ( n = 26) and Arl8b siRNA ( n = 66) in wound-healing assay (µm/h, mean of cell migration speeds ± SD). (F) Colocalization of Paxillin and Rab7 in MEFs. Images from time-lapse series of MEF cells coexpressing GFP-Rab7 (green) and mCherry-Paxillin (red). White arrows indicate FAs targeted by GFP-Rab7. See also Video 10 .

Article Snippet: The mouse monoclonal anti-Paxillin antibody was bought from EMD Millipore, and the rat anti-LAMP1 (CD107a) antibody was purchased from Merck.

Techniques: Expressing, Transfection, Migration, Wound Healing Assay

Journal: The Journal of Cell Biology

Article Title: The late endosomal p14–MP1 (LAMTOR2/3) complex regulates focal adhesion dynamics during cell migration

doi: 10.1083/jcb.201310043

Figure Lengend Snippet: Absence of p14–MP1 or blockage of Arl8b-dependent late endosomal transport causes IQGAP1 accumulation in FAs. (A) IF: anti-Paxillin and anti-IQGAP1 antibodies. IQGAP1 localizes to the leading edge in p14 f/− (white arrows) and colocalizes with Paxillin in FAs in p14 −/− MEFs (red arrows). (B) IF: anti-Paxillin and anti-IQGAP1 antibodies. Shown are different time points during spreading of p14 f/− and p14 −/− MEFs. White arrows indicate accumulation of IQGAP1 at the leading edge in control MEFs. Red arrows point at IQGAP1 localization at Paxillin-positive FAs in control and p14 −/− MEFs. (C) IF: anti-LAMP1 (green), anti-IQGAP1 (red) antibodies, and Hoechst (blue). IQGAP1 localizes to the leading edge in p14 f/− MEFs treated with control RNAi (white arrows) and localizes to FAs upon Arl8b depletion (red arrows). (D) p14 f/− MEFs treated as in C. IF: anti-Paxillin and anti-IQGAP1 antibodies. Note IQGAP1 localization to the leading edge in p14 f/− MEFs treated with control RNAi (white arrows) versus colocalization of IQGAP1 and Paxillin in FAs upon ARl8b depletion (red arrows).

Article Snippet: The mouse monoclonal anti-Paxillin antibody was bought from EMD Millipore, and the rat anti-LAMP1 (CD107a) antibody was purchased from Merck.

Techniques: