Journal: FEBS Open Bio

Article Title: Inactivation of the three GGA genes in HeLa cells partially compromises lysosomal enzyme sorting

doi: 10.1002/2211-5463.13040

Figure Lengend Snippet: Sorting of cathepsin D in the absence of GGA proteins. (A) Parental WT, GGA1 −/− , GGA2 −/− , GGA3 −/− , and GGA123 −/− HeLa cell lines were metabolically labeled with [ 35 S]methionine/cysteine and processed as described under ‘ ’. Secreted and intracellular cathepsin D molecules were immunoprecipitated with a polyclonal anti‐cathepsin D antibody, resolved by 10% SDS/PAGE and visualized by autoradiography of the dried gel. A representative autoradiograph is shown. (B) The percentage of cathepsin D in the media (m) was calculated as the ratio of radioactivity in the secreted form divided by the sum of the processed (c) and secreted forms (m). The data shown are the mean ± SD for four independent experiments. (C) Parental WT, GGA123 −/− , and GNPTAB −/− HeLa cell lines were processed for cathepsin D sorting as described in (A). A representative autoradiograph is shown, and data presented are the mean of two independent experiments.

Article Snippet: The following published/validated commercial antibodies against human GGA proteins were used to screen for the GGA knockout cell lines: anti‐GGA1 rabbit monoclonal (Abcam, Cat # ab170956, Cambridge, MA, USA), anti‐GGA2 mouse monoclonal (BD Biosciences, Cat # 612612), and anti‐GGA3 rabbit monoclonal (Cell Signaling, Cat # D66F1, Beverly, MA, USA).

Techniques: Metabolic Labelling, Labeling, Immunoprecipitation, SDS Page, Autoradiography, Radioactivity

Journal: EMBO Molecular Medicine

Article Title: An aberrant sugar modification of BACE1 blocks its lysosomal targeting in Alzheimer's disease

doi: 10.15252/emmm.201404438

Figure Lengend Snippet: Down-regulation of BACE1 protein in aged Mgat3 −/− mouse brain Immunostaining of 12-month-old brain sections (cerebral cortex) with anti-BACE1. Scale bar, 100 μm. Proteins from the membrane fractions of 12-month-old mouse brains ( n = 4–5) were immunoblotted for BACE1, and BACE1 intensity was quantified. * P = 0.044. Lysates from MEFs were immunoblotted for APP, BACE1, or GAPDH (loading control), and BACE1 intensity was quantified ( n = 4). Amount of Aβ40 in the culture medium of immortalized MEFs ( n = 3). * P = 0.045. MEFs were transfected with control siRNA or GGA3-siRNA and then immunoblotted for BACE1, GGA3, or GAPDH (loading control) ( n = 6). Data Information: All graphs show means ± SEM (* P < 0.05; Student's t -test for (B) and the Mann–Whitney U -test for (D)). Source data are available online for this figure.

Article Snippet: The commercially available antibodies used were as follows: rabbit anti-BACE1 (5606), anti-nicastrin (9447S), anti-GGA3 (8027), anti-rab5 (3547), anti-rab7 (9367), and anti-rab9 (5118) from Cell Signaling Technology; anti-APP C-term (recognizes C-terminal part of APP, 18961), anti-APP N-term (10D1), and anti-sAPPβ-sw (10321, clone 6A1) from Immuno-Biological Laboratories; anti-Aβ (SIG-39220, clone 4G8) and anti-sAPPα (SIG-39320, clone 6E10) from SIGNET; anti-Lamp1 (ab25630) and anti-PSD95 (ab2723) from Abcam; anti-actin (A4700) from Sigma; anti-syntaxin 6 (610635) from BD Biosciences; anti-GAPDH (MAB374), anti-APP (22C11), and anti-MBP (MAB386) from Millipore; anti-MAP2 (sc-20172) from Santa Cruz Biotechnology; anti-GFAP (13-0300) from Life Technologies; anti-CHL1 (AF2147) and anti-contactin-2 (AF4439) from R&D systems; and anti-Iba1 (019-197419) from Wako.

Techniques: Immunostaining, Transfection, MANN-WHITNEY

Journal: The Journal of Biological Chemistry

Article Title: Sequential Activation and Inactivation of Dishevelled in the Wnt/?-Catenin Pathway by Casein Kinases

doi: 10.1074/jbc.M110.169870

Figure Lengend Snippet: TABLE 1

Article Snippet: The antibodies used include phospho-LRP6 (Ser-1490, #2568), Dvl3 (#3218), Dvl2 (#3224), Axin1 (#2087) from Cell Signaling Technologies, actin (C-11, sc-1615), Dvl3 (sc-8027), Dvl2 (sc-8026), CK1ϵ (sc-6471), and Myc (sc-40) from Santa Cruz Biotechnology, CK2α (#611610) from BD Transduction Laboratories, and FLAG M2 (F1804) from Sigma.

Techniques: