rabbit polyclonal anti human lamp1  (Novus Biologicals)

Journal: PLoS ONE

Article Title: Role of SNX16 in the Dynamics of Tubulo-Cisternal Membrane Domains of Late Endosomes

doi: 10.1371/journal.pone.0021771

Figure Lengend Snippet: A ) HeLa cells were transfected with Venus-SNX16 and analyzed by immunofluorescence microscopy using antibodies against LAMP1. B ) HeLa cells co-expressing mRFP-SNX16 and EGFP-RAB7 were fixed and analyzed by fluorescence microscopy (see also ). C ) HeLa cells co-expressing mRFP-SNX16 and EGFP-RILP were analyzed by fluorescence video microscopy (see ). D ) HeLa cells transfected with Venus-SNX16 were analyzed by immunofluorescence microscopy using antibodies against LBPA. E ) HeLa cells were co-transfected with Venus-SNX16 and CD63-mRFP and analyzed by fluorescence video microscopy.

Article Snippet: We also used mouse monoclonal antibodies against transferrin receptor (Zymed Laboratories, South San Francisco, CA), rabbit polyclonal anti-EEA1 (Enzo Life Sciences, Plymouth Meeting, PA), mouse monoclonal anti-EEA1 (BD Biosciences, Franklin Lakes, NJ), mouse monoclonal anti-human LAMP1 (CD107a; BD Biosciences) and rabbit polyclonal anti-human LAMP1 (Thermo Fisher Scientific, Waltham, MA).

Techniques: Transfection, Immunofluorescence, Microscopy, Expressing, Fluorescence

Journal: PLoS ONE

Article Title: Role of SNX16 in the Dynamics of Tubulo-Cisternal Membrane Domains of Late Endosomes

doi: 10.1371/journal.pone.0021771

Figure Lengend Snippet: A–B ) HeLa cells transfected with Venus-SNX16 were labeled with antibodies against LAMP1 and LBPA (A) or LAMP1 and CD63 (B), and analyzed by confocal microscopy. The distribution of Venus-SNX16 under low expression conditions, LAMP1, and LBPA (A) or Venus-SNX16, LAMP1, and CD63 (B) was quantified after 3D image reconstruction using Imaris software (error bars indicate STDEVA). The data are expressed as the percentage of LAMP1, which co-distributes with the indicated marker. C–D ) Untransfected BHK cells were homogenized and a post-nuclear supernatant (PNS) was prepared. The PNS was fractionated by floatation using a well-established step sucrose gradient . Early (EE) and late (LE) endosome fractions were collected and analyzed by SDS gel electrophoresis and western blotting with antibodies against LAMP1, SNX16 or RAB5, or by ELISA with antibodies against LBPA. In (C), the gels were loaded with equal amounts of protein (2.5 µg), as were the wells in the ELISA analysis (5 µg), to visualize enrichment of the corresponding markers in the fractions. RFU: relative fluorescence units. In (D), the gels were loaded with equal volume (1/3 of the total fraction) to visualize the yields of the corresponding markers in the fractions. In the LBPA analysis, yields were calculated from the quantification of the ELISA data (total RFU).

Article Snippet: We also used mouse monoclonal antibodies against transferrin receptor (Zymed Laboratories, South San Francisco, CA), rabbit polyclonal anti-EEA1 (Enzo Life Sciences, Plymouth Meeting, PA), mouse monoclonal anti-EEA1 (BD Biosciences, Franklin Lakes, NJ), mouse monoclonal anti-human LAMP1 (CD107a; BD Biosciences) and rabbit polyclonal anti-human LAMP1 (Thermo Fisher Scientific, Waltham, MA).

Techniques: Transfection, Labeling, Confocal Microscopy, Expressing, Software, Marker, SDS-Gel, Electrophoresis, Western Blot, Enzyme-linked Immunosorbent Assay, Fluorescence

Journal: PLoS ONE

Article Title: Role of SNX16 in the Dynamics of Tubulo-Cisternal Membrane Domains of Late Endosomes

doi: 10.1371/journal.pone.0021771

Figure Lengend Snippet: A–B ) HeLa cells transfected with Venus-SNX16 were fixed with paraformaldehyde (A) or glutaraldehyde (0.3%) and paraformaldehyde (3%) for 50 min (B) and analyzed by immunofluorescence microscopy using antibodies against LAMP1. Green arrows point at Venus-SNX16-positive tubules without detectable LAMP1, white arrows point to LAMP1- and SNX16-containing tubules. C ) The left panel shows a confocal section of a cell expressing Venus-SNX16 and labeled for LAMP1 (fixation as in B). The middle panel shows a 3D reconstruction of the corresponding confocal stack with Imaris software, and the right panel shows a magnification of the boxed region, displaying only Venus-SNX16 and its colocalization with LAMP1. White arrows point out the presence of LAMP1 at discrete sites of Venus-SNX16-decorated tubules. D ) HeLa cells transfected with Venus-SNX16 were treated with brefeldin A (5 µg/ml for 30 min) prior to fixation with paraformaldehyde and analyzed by immunofluorescence microscopy using antibodies against TFR and the cis -Golgi protein p23. The insert in the p23 panel shows the characteristic ribbon-like distribution of p23 in control cells without brefeldin A.

Article Snippet: We also used mouse monoclonal antibodies against transferrin receptor (Zymed Laboratories, South San Francisco, CA), rabbit polyclonal anti-EEA1 (Enzo Life Sciences, Plymouth Meeting, PA), mouse monoclonal anti-EEA1 (BD Biosciences, Franklin Lakes, NJ), mouse monoclonal anti-human LAMP1 (CD107a; BD Biosciences) and rabbit polyclonal anti-human LAMP1 (Thermo Fisher Scientific, Waltham, MA).

Techniques: Transfection, Immunofluorescence, Microscopy, Expressing, Labeling, Software

Journal: PLoS ONE

Article Title: Role of SNX16 in the Dynamics of Tubulo-Cisternal Membrane Domains of Late Endosomes

doi: 10.1371/journal.pone.0021771

Figure Lengend Snippet: A–F ) HeLa cells co-transfected with Venus-SNX16 and HRP-LAMP1 were processed as described in , . Briefly, cells were chased with 1 mM DDT for 30 min, to ensure proper HRP-LAMP1 localization . Prior to fixation, HRP-LAMP1 was revealed cytochemically with the DAB reaction and cells were permeabilized; each treatment was for 30 min at 4°C under physiological osmolarity conditions . Samples were analyzed by phase contrast microscopy to reveal HRP-LAMP1 (A) and by fluorescence microscopy to reveal Venus-SNX16 (B). Panel C shows the merged image of A) and B), and panel D a high magnification view of the region boxed in C). In E), an example of a cell is shown where Venus-SNX16 and HRP-LAMP1 colocalize on numerous tubules (magnification in F).

Article Snippet: We also used mouse monoclonal antibodies against transferrin receptor (Zymed Laboratories, South San Francisco, CA), rabbit polyclonal anti-EEA1 (Enzo Life Sciences, Plymouth Meeting, PA), mouse monoclonal anti-EEA1 (BD Biosciences, Franklin Lakes, NJ), mouse monoclonal anti-human LAMP1 (CD107a; BD Biosciences) and rabbit polyclonal anti-human LAMP1 (Thermo Fisher Scientific, Waltham, MA).

Techniques: Transfection, Microscopy, Fluorescence

Journal: PLoS ONE

Article Title: Role of SNX16 in the Dynamics of Tubulo-Cisternal Membrane Domains of Late Endosomes

doi: 10.1371/journal.pone.0021771

Figure Lengend Snippet: A ) HeLa cells transfected with Venus-SNX16 were treated or not with 10 µM nocodazole to depolymerize the microtubules as in , fixed in 0.3% glutaraldehyde and 3% paraformaldehyde, and analyzed by immunofluorescence microscopy using antibodies against LAMP1. B ) HeLa cells overexpressing Venus-SNX16 were analyzed by immunofluorescence microscopy using antibodies against LBPA. C ) BHK cells treated or not with nocodazole as in (A) were fractionated as in . Early (EE) and late (LE) endosome fractions were analyzed by SDS gel electrophoresis and western blotting using the indicated antibodies. Gels were loaded with equal amounts of protein. D ) BHK cells overexpressing myc-SNX16 were fractionated and analyzed as in (C). E ) EGFP-SNX16-overexpressing cells were processed for cryosectioning and labeled with anti-GFP antibodies, as described , . Scale bar is 100 nm.

Article Snippet: We also used mouse monoclonal antibodies against transferrin receptor (Zymed Laboratories, South San Francisco, CA), rabbit polyclonal anti-EEA1 (Enzo Life Sciences, Plymouth Meeting, PA), mouse monoclonal anti-EEA1 (BD Biosciences, Franklin Lakes, NJ), mouse monoclonal anti-human LAMP1 (CD107a; BD Biosciences) and rabbit polyclonal anti-human LAMP1 (Thermo Fisher Scientific, Waltham, MA).

Techniques: Transfection, Immunofluorescence, Microscopy, SDS-Gel, Electrophoresis, Western Blot, Labeling