Journal: Frontiers in Molecular Biosciences

Article Title: Primary cilia-mediated regulation of microglial secretion in Alzheimer’s disease

doi: 10.3389/fmolb.2023.1250335

Figure Lengend Snippet: Information of antibodies used in figures.

Article Snippet: . , Anti-Lamp1 (1D4B) (1:100) , Developmental Studies Hybridoma Bank (DSHB) , 1D4B , Rat , Donkey anti-Rat IgG (H + L) Secondary Antibody, Alexa Fluor ® 594 conjugate (1:250) , Life technologies , A21209.

Techniques: Staining

Journal:

Article Title: Zinc transporter ZIP8 (SLC39A8) and zinc influence IFN-? expression in activated human T cells

doi: 10.1189/jlb.1208759

Figure Lengend Snippet: ZIP8 protein is up-regulated in response to primary T cell activation and localizes with lysosomes. Western blot analysis (A and B) and confocal images (C and D) from primary T cells. Total membrane proteins and total cell lysates were isolated and used for SDS-PAGE. Blots were probed with an affinity-purified polyclonal hZIP8 antibody. (A) Blots were probed with affinity-purified, polyclonal hZIP8, directly or after preincubation with its peptide antigen (Pept). Act, Activated cell. (B) Representative Western blots from three independent experiments for membrane proteins and total cell lysates. The average values of both ZIP8 band intensities from three independent experiments are shown. Values shown are means ± sd (n=6); *, P < 0.02: **, P < 0.001, compared with nonactivated control (Cont). (C) Immunofluororescence localization of ZIP8 (red) by laser-scanning confocal microscopy. Nonactivated and activated primary T cells probed with the ZIP8 antibody. The area in white boxes represents magnified images. (D) Immunofluororescence localization of ZIP8 (red) and LAMP1 (green), a lysosome-specific membrane marker, in activated T cells by laser-scanning confocal microscopy. The area that was chosen for enlarged images was indicated by white squares. Yellow indicates the extent of colocalization.

Article Snippet: Immunolabeling was with the anti-hZIP8 polyclonal antibody or goat anti-LAMP1 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Activation Assay, Western Blot, Isolation, SDS Page, Affinity Purification, Confocal Microscopy, Marker

Journal: Journal of autoimmunity

Article Title: Intracellular trafficking of beta2-glycoprotein I complexes with lipid vesicles in macrophages: implications on the development of antiphospholipid syndrome.

doi: 10.1016/j.jaut.2007.07.003

Figure Lengend Snippet: Fig. 2. Intracellular trafficking of b2GPI complexed with lipid vesicles. (A) Internalization of preformed oxLDL/b2GPI complexes and the accumulation of lipids in the J774 cells. Cells were incubated with the preformed oxLDL/‘‘labeled b2GPI’’ complexes (labeled with Alexa488; as described in Section 2) at 37 C for 30 min, 4 h, and 8 h. The green and red fluorescence come from the Alexa488-labeled b2GPI and the Nile-red-stained lipid droplets, respectively. (B) The cells were incubated with oxLDL/’’labeled b2GPI’’ complexes at 37 C for 8 h. The data indicate that oxLDL/b2GPI complexes were co-localized with LAMP1 but not with EEA1 or Rab 7 after incubation for 8 h. (C) Green fluorescent Alexa488-labeled b2GPI was merged with LAMP1 after incubating with PS-liposomes at 37 C for 16 h. The white asterisk (*) indicates the location of the nuclei and the yellow bar indicates a length of 10 mm.

Article Snippet: Chemicals and antibodies 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dipalmitoyl-sn-glycero-3-[phospho-L-serine] (DPPS) were purchased from Avanti Polar Lipids Inc. (Alabaster, AL); Nile red from SigmaeAldrich Co. (St. Louis, MO); rabbit IgG anti-EEA1 from Upstate Cell Signaling (Lake Placid, NY); rabbit IgG anti-mouse Rab7 from Santa Cruz Biotechnology Inc. (Santa Cruz, CA); rat IgG anti-mouse LAMP1 from Southern Biotechnology Associates Inc. (Birmingham, AL); mouse IgA anti-mouse CD36 from BD Biosciences (San Jose, CA); rat IgG anti-mouse FcgRI/CD64 from R&D Systems Inc. (Minneapolis, MN); FITC-labeled goat IgG anti-mouse IgA from AbD Serotec (Oxford, UK); FITClabeled goat IgG anti-mouse IgG from Zymed Laboratories (South San Francisco, CA); LysoTracker, Alexa Fluor 546 (Alexa546 with red fluorescence) labeled goat IgG anti-rabbit IgG, and goat IgG anti-rat IgG from Molecular Probes (Eugene, OR).

Techniques: Incubation, Labeling, Staining, Liposomes

Journal: Journal of autoimmunity

Article Title: Intracellular trafficking of beta2-glycoprotein I complexes with lipid vesicles in macrophages: implications on the development of antiphospholipid syndrome.

doi: 10.1016/j.jaut.2007.07.003

Figure Lengend Snippet: Fig. 3. IgG anti-b2GPI-dependent uptake of b2GPI complexed with lipid vesicles. (A) The internalization of b2GPI/oxLDL complexes began to be detected in the intracellular spaces in the presence of WB-CAL-1 after incubation for 30 min at 37 C and the complexes were merged with lipid droplets. (B) Co-localization profiles of the green fluorescence of ‘‘labeled b2GPI’’/oxLDL were partly merged with all three organelle markers (i.e., EEA1, Rab7, and LAMP1) after 30 min. In contrast, most of the green fluorescence was merged only with LAMP1 after 8 h. (C) Similar co-localization profiles of blue fluorescence Alexa488-labeled b2GPI are observed after incubating with a free form of Alexa488 labeled-b2GPI, PS-liposomes, and WB-CAL-1 for 30 min or 8 h at 37 C. The white asterisk (*) indicates the location of the nuclei and the yellow bar indicates a length of 10 mm.

Article Snippet: Chemicals and antibodies 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dipalmitoyl-sn-glycero-3-[phospho-L-serine] (DPPS) were purchased from Avanti Polar Lipids Inc. (Alabaster, AL); Nile red from SigmaeAldrich Co. (St. Louis, MO); rabbit IgG anti-EEA1 from Upstate Cell Signaling (Lake Placid, NY); rabbit IgG anti-mouse Rab7 from Santa Cruz Biotechnology Inc. (Santa Cruz, CA); rat IgG anti-mouse LAMP1 from Southern Biotechnology Associates Inc. (Birmingham, AL); mouse IgA anti-mouse CD36 from BD Biosciences (San Jose, CA); rat IgG anti-mouse FcgRI/CD64 from R&D Systems Inc. (Minneapolis, MN); FITC-labeled goat IgG anti-mouse IgA from AbD Serotec (Oxford, UK); FITClabeled goat IgG anti-mouse IgG from Zymed Laboratories (South San Francisco, CA); LysoTracker, Alexa Fluor 546 (Alexa546 with red fluorescence) labeled goat IgG anti-rabbit IgG, and goat IgG anti-rat IgG from Molecular Probes (Eugene, OR).

Techniques: Incubation, Labeling, Liposomes

Journal: Frontiers in Pharmacology

Article Title: Molecular Mechanisms of Epileptic Encephalopathy Caused by KCNMA1 Loss-of-Function Mutations

doi: 10.3389/fphar.2021.775328

Figure Lengend Snippet: Autophagy in BK KO mice was abnormal. Calcium imaging found that the activation of BK channel could activate lysosomal trpml1 (autophagy key calcium channel). (A) Co labeling of microglia, LC3B, IBA-1, and LAMP1 in control mice. (B) Co labeling of microglia, LC3B, IBA-1, and LAMP1 in BK KO mice. (C) NS1619 was applied to HEK293T transfected with BK channel and TRPML1-GCaMP3 in order to detect its regulation of lysosomal calcium outflow ( ∗∗ p < 0.01, n = 6).

Article Snippet: These include against LC3B (1:200 dilution; ab48394; Abcam), LAMP1(1:500 dilution; ab25630; Abcam), and Iba-1 (1:500 dilution; ab178846; Abcam). washing four times in PBS (4 × 5 min), sections were incubated with goat anti-rabbit antibody conjugated with Alexa Fluor 594 (for LC3B; 1:200 dilution; ab150080; Abcam); goat polyclonal secondary antibody to mouse conjugated with Alexa Fluor 488 (for LAMP1; 1:200 dilution; ab150113; Abcam) and Alexa Fluor 350-labeled goat anti-rabbit IgG (H + L) (for Iba-1; 1:500 dilution; A0408; Beyotime) for 1 h at RT, washing four times in PBS (4 × 5 min).

Techniques: Imaging, Activation Assay, Labeling, Transfection

Journal: Cell Reports

Article Title: Modeling Glycan Processing Reveals Golgi-Enzyme Homeostasis upon Trafficking Defects and Cellular Differentiation

doi: 10.1016/j.celrep.2019.03.107

Figure Lengend Snippet:

Article Snippet: Primary antibodies used in this study were anti-LAMP1 (1/100, gift from Paul Pryor, University of York), anti-GalT (1/500, R&D Systems, AF3609), anti-MGAT1 (1/500, Abcam, ab180578), anti-FUT8 (1/1000, Abcam, ab191571) and anti-GAPDH (1/500,000, Applied Biosystems).

Techniques: Recombinant, Imaging, Western Blot, Knockdown, Knock-Out, Software, Transfection