Journal: Frontiers in Immunology

Article Title: Investigating the role and regulation of GPNMB in progranulin-deficient macrophages

doi: 10.3389/fimmu.2024.1417836

Figure Lengend Snippet: List of qPCR primers for genes of interest.

Article Snippet: Ctsd (Origene, NM_009983) , TAA GAC CAC GGA GCC AGT GTC A , CCA CAG GTT AGA GGA GCC AGT A.

Techniques:

Journal: The Journal of Cell Biology

Article Title: Fam20C regulates protein secretion by Cab45 phosphorylation

doi: 10.1083/jcb.201910089

Figure Lengend Snippet: Sorting of non-Cab45 cargos is not affected in Cab45 phosphomutants (related to ). (A) Western blot analysis of the secretion of CatD in HeLa control and Fam20C-KO cells. Endogenous CatD was trapped in the Golgi at 20°C and released for 1 h at 37°C. The supernatants (upper panel) and cell lysates (lower panels) were tested for CatD by Western blotting. β-Actin was used as loading control. (B) Representative immunofluorescence images of colocalization of vesicular SBP-EGFP-CatD with the lysosomal marker Lamp1. HeLa cells were transfected with RUSH construct and incubated for 40 min with biotin. Cells were fixed and stained with α-Lamp1 antibody. Scale bar, 10 µm. The magnification of the inset is shown in the lower right pane (scale bar, 1 µm). (C) Representative immunofluorescence images of RUSH experiments showing OPN transport in HeLa control, Cab45-KO, Cab45-WT, and Cab45 phosphomutant cell lines. Cells were transfected with OPN-SBP-EGFP and fixed at 0, 20, 40, and 50 min after the addition of biotin. Z-stack images (d = 0.35 µm) were analyzed. The arrowheads indicate cytoplasmic vesicles. Scale bars, 10 µm. (D) Representative immunofluorescence images of RUSH experiments showing cathepsin D (CatD) transport in Cab45-WT and phosphomutant cell lines. Cells were transfected with SBP-EGFP-CatD and fixed at 0, 20, 40, and 60 min after the addition of biotin. Z-stack images (d = 0.35 µm) were analyzed. The arrowheads indicate cytoplasmic vesicles. Scale bars, 10 µm. (E) The numbers of CatD budding vesicles (D) were quantified. The cytoplasmic vesicles were counted at each time point by analyzing z-stack images (d = 0.35 µm). Scatter dot plot represents the means ± SD of at least three independent experiments ( n > 30 cells per condition). Statistical test, Kruskal–Wallis.

Article Snippet: The antisera used were rat monoclonal anti-HA (Roche; catalog #11867423001), rabbit monoclonal anti-KIAA0310 (Thermo Fisher Scientific; catalog #A300-648A-M), mouse monoclonal anti-cis-Golgi matrix protein 130 (BD Biosciences; catalog #610823), mouse monoclonal anti-CNX (Sigma-Aldrich; catalog #C7617), mouse monoclonal anti-p230 (BD Biosciences; catalog #611281), sheep polyclonal anti-TGN46 (AbD Serotec; catalog #AHP500G), mouse monoclonal anti-β-actin (Sigma-Aldrich; catalog #A5441), mouse monoclonal ANTI-FLAG M2-peroxidase (HRP; Sigma-Aldrich; catalog #A8592), mouse monoclonal anti-EEA1 antibody (BD Biosciences; catalog #610457), rabbit polyclonal anti-Rab11 antibody (gift from the De Camilli laboratory, Yale School of Medicine, New Haven, CT, self-made), rabbit monoclonal anti-Lamp1 antibody (Cell Signaling; catalog #9091S), and mouse monoclonal anti-CatD antibody (BD Biosciences; catalog #610801).

Techniques: Western Blot, Immunofluorescence, Marker, Transfection, Construct, Incubation, Staining

Journal: Alzheimer's Research & Therapy

Article Title: Prominent tauopathy and intracellular β-amyloid accumulation triggered by genetic deletion of cathepsin D: implications for Alzheimer disease pathogenesis

doi: 10.1186/s13195-024-01443-6

Figure Lengend Snippet: Inhibition of CatD slows the catabolism of tau in vitro and in cultured cells. ( A ) Representative Coomassie blue-stained polyacrylamide gel loaded with recombinant human tau (rTau) incubated for the indicated times with recombinant human CatD (5 nM) in the absence or presence of equal concentrations (1 µM) of Aβ40 or Aβ42. ( B ) Quantification of rTau levels as a function of time in 4 independent experiments. Note how rTau catabolism is unaffected by Aβ40 but markedly slowed by Aβ42, a potent competitive inhibitor of CatD. Data are mean ± SEM; n = 4. ( C ) Overview of the experimental approach used to quantify hTau catabolism in “Tet-Off” cultured neuroblastoma cells (see main text). ( D ) Representative western blot showing hTau levels (stained with antibody P44) at different time points after cessation of hTau expression in the absence or presence of the CatD inhibitor, pepstatin A (PepA; 1 µM). ( E ) Quantitation of hTau levels as a function of time from 6 independent experiments. Note the marked increase in the half-life of hTau in the presence of PepA (0.98 days; 95% CI 0.80 to 1.25) relative to DMSO-treated controls (0.51 days; 95% CI 0.429 to 0.627; P = 0.0012). Data are mean ± SEM, n = 6

Article Snippet: Reactions were initiated by addition of purified human CatD (2.5 nM; Enzo Life Sciences, Farmingdale, NY) dissolved in Assay Buffer, then 20-µL aliquots were removed 0, 0.5, 1, 2, and 4 h thereafter, with CatD activity in each aliquot immediately terminated by addition of PepA (1 µM) and incubation on ice.

Techniques: Inhibition, In Vitro, Cell Culture, Staining, Recombinant, Incubation, Western Blot, Expressing, Quantitation Assay