Journal: Biochemical Journal

Article Title: Deciphering the LRRK code: LRRK1 and LRRK2 phosphorylate distinct Rab proteins and are regulated by diverse mechanisms

doi: 10.1042/BCJ20200937

Figure Lengend Snippet: ( A ) Wild type, and homozygous VPS35[D620N] knock-in primary MEFs were deprived of serum for 16 h and then incubated in the presence or absence of 100 ng/ml PMA for 30 min. Extracts (20 µg) from triplicate independent clones were subjected to immunoblot analysis with the indicated antibodies (all at 1 µg/ml). Each lane represents cell extract obtained from a different cell clone. The membranes were developed using the LI-COR Odyssey CLx Western Blot imaging system. Similar results were obtained in three independent experiments. ( B , upper) HeLa cells were transfected with wild type GFP-LRRK1 or GFP-LRRK2 in the presence of absence of HA-Rab29. Twenty-four hours post-transfection cells were fixed in 4% (by vol) paraformaldehyde and stained with mouse anti-HA. Scale bar represents 30 µm. ( B , lower) As in ( B , upper) except that cell are lysed and analyzed by immunoblot analysis as described in ( A ).

Article Snippet: Rabbit monoclonal MJFF-pRab10 (Thr73) (#ab230261, Abcam, Inc. [ ]), rabbit monoclonal MJFF-pRab8 (Thr72) (#ab230260, Abcam, Inc. [ ]), mouse monoclonal MJFF-total Rab10 antibody (#0680-100/Rab10-605B11, nanotools [ ]), mouse monoclonal anti-LRRK2 (#75-253, NeuroMab), mouse monoclonal anti-Rab8A (#WH0004218M2, Sigma–Aldrich), mouse anti-α-Tubulin (#2144, Cell Signaling Technologies (CST)), rabbit polyclonal pERK1/2 (T202, Y204) (#9101, CST), rabbit monoclonal pEGFR (Y1173) (#4407, CST), rabbit monoclonal pAMPK (Thr172) (#4188, CST), mouse monoclonal total AMPK (CST, #2793), rabbit polyclonal pACC (Ser79) (CST, #3661), rabbit monoclonal pVASP (Ser157) (#84519, CST), rabbit polyclonal p-Ser PKC Substrate (#2261, CST), rabbit monoclonal pTBK1 (Ser172) (#5483, CST), rabbit polyclonal total TBK1 (#3013, CST), rabbit monoclonal pP105 (Ser933) (#4806, CST), rabbit monoclonal pP38 (Thr180, Tyr182) (#4631, CST), mouse monoclonal anti-Total Ub (#646302, BioLegend), mouse monoclonal anti-OPA-1 (#612607, BD Biosciences), mouse anti-GAPDH (#sc-32233, Santa Cruz Biotechnology), and rabbit anti-GFP (#PABG1, Chromotek), Rat anti-HA (#ROAHAHA, Sigma), mouse monoclonal anti-Rab7A (#R8779, Sigma), rabbit polyclonal anti-pAkt (Ser473) (#9271, CST), mouse monoclonal VPS35 (#SMC-605D, StressMarq Biosciences).

Techniques: Knock-In, Incubation, Clone Assay, Western Blot, Imaging, Transfection, Staining

Journal: Nature Communications

Article Title: CCC- and WASH-mediated endosomal sorting of LDLR is required for normal clearance of circulating LDL

doi: 10.1038/ncomms10961

Figure Lengend Snippet: ( a ) Human embryonic kidney 293T (HEK293T) cells were transfected with constructs expressing Flag-LDLR with either COMMD1-GST or GST alone. Interaction with COMMD1 was detected via pull-down assay using glutathione sepharose beads. ( b ) HEK293T cells were transfected with Flag-LDLR vector, and interaction with endogenous COMMD1 was detected by immunoprecipitation with rabbit anti-Flag-antibody. ( c ) Colocalization of LDLR (red) and COMMD1 (green) in Commd1 f/f MEFs examined by immunofluorescence staining. Representative images are shown; scale bar, 5μm. LDLR (red) and COMMD1 (green) was stained in COMMD1-deficient MEFs ( Commd1 −/− ) and imaged by confocal fluorescence microscopy. COMMD1 levels in Commd1 f/f and in Commd1 −/− MEFs determined by immunoblot analysis. ( d ) Liver of a WT chow-fed mouse was homogenized and loaded on a continuous 10–40% sucrose gradient. Fractions were separated by ultracentrifugation and immunoblotted using antibodies against COMMD1, LDLR, WASH1, FAM21, VPS35 and CCDC22. The figure represents results of three independent experiments. ( e ) HEK293T cells were transfected with Ha-COMMD1 construct together with GST alone, GST-LDLRct (GST-tagged cytosolic domain of LDLR) or GST-LDLRct Y807A (GST-tagged mutated cytosolic domain of LDLR). Pull-down assay was performed to study the interaction between LDLRct and COMMD1. ( f ) Lysates of Flag-LDLR-transfected HEK293T cells were used for immunoprecipitation assays. Immunoprecipitates were washed, separated by SDS–polyacrylamide gel electrophoresis and immunoblotted as indicated.

Article Snippet: The following antibodies were used: rabbit polyclonal antibody against LDLR (PAB8804, Abnova, 1:200); mouse monoclonal antibody against COMMD1 (MAB7526, R&D Systems, 1:400); rabbit polyclonal antibody against EEA1 (2411, Cell Signaling Technologies, 1:250); goat polyclonal antibody against VPS35 (ab10099-100, Abcam, 1:1,000); rat monoclonal antibody against LAMP1 (553792, BD Biosciences, 1:500); rabbit polyclonal antibody against WASH1 (1:1,000); and rabbit polyclonal antibody against FAM21 (1:1,000) (ref. ).

Techniques: Transfection, Construct, Expressing, Pull Down Assay, Plasmid Preparation, Immunoprecipitation, Immunofluorescence, Staining, Fluorescence, Microscopy, Western Blot, Polyacrylamide Gel Electrophoresis

Journal: Nature Communications

Article Title: CCC- and WASH-mediated endosomal sorting of LDLR is required for normal clearance of circulating LDL

doi: 10.1038/ncomms10961

Figure Lengend Snippet: ( a ) LDLR (green), COMMD1 (red) and VPS35 (pink) were stained in Commd1 f/f and Commd1 −/− MEFs and imaged by confocal microscopy. Representative images are shown; scale bar, 5μm. ( b ) Quantification of the colocalization of LDLR with COMMD1, VPS35, EEA1 and LAMP1 was performed by the analysis of 30–40 cells. ( c ) Total and plasma membrane LDLR levels of Commd1 f/f and Commd1 −/− MEFs determined by biotinylation assay. Data represent three independent experiments, and ( d ) the relative levels of LDLR at the cell surface are quantified in all experiments. ( e ) In vitro LDL and transferrin uptake assay. Dil-labelled LDL (5 μg ml −1 ) or Alexa-633-labelled transferrin (5 μg ml −1 ) was added to serum-depleted medium and incubated with MEFs at 4 °C for 1 h and subsequently at 37 °C for 5 min. Dil-labelled LDL and Alexa-633-labelled transferrin uptake was measured by FACS analysis, and the relative uptake in triplicate is shown. The results are presented as mean±s.e.m.; significance was calculated relative to the control group by unpaired Student's t -test; * P <0.05, *** P <0.001.

Article Snippet: The following antibodies were used: rabbit polyclonal antibody against LDLR (PAB8804, Abnova, 1:200); mouse monoclonal antibody against COMMD1 (MAB7526, R&D Systems, 1:400); rabbit polyclonal antibody against EEA1 (2411, Cell Signaling Technologies, 1:250); goat polyclonal antibody against VPS35 (ab10099-100, Abcam, 1:1,000); rat monoclonal antibody against LAMP1 (553792, BD Biosciences, 1:500); rabbit polyclonal antibody against WASH1 (1:1,000); and rabbit polyclonal antibody against FAM21 (1:1,000) (ref. ).

Techniques: Staining, Confocal Microscopy, Clinical Proteomics, Membrane, Cell Surface Biotinylation Assay, In Vitro, Incubation, Control

Journal: Nature Communications

Article Title: CCC- and WASH-mediated endosomal sorting of LDLR is required for normal clearance of circulating LDL

doi: 10.1038/ncomms10961

Figure Lengend Snippet: ( a ) Cellular localization of LDLR (green), COMMD1 (red) and LAMP1 (pink) in Wash1 f/f and Wash1 −/− MEFs was determined by immunofluorescence staining. Representative images are shown; scale bar, 5 μm. Relative colocalization of ( b ) LDLR and ( c ) COMMD1 with VPS35, EEA1 and LAMP1 was quantified. ( d ) LDLR, WASH1, VPS35 and COMMD1 levels in Wash1 f/f and Wash1 −/− MEFs analysed by western blot. ( e ) Representative images ( n =3) of immunoblot analysis of total LDLR levels in Wash1 f/f and Wash1 −/− MEFs treated with bafilomycin A (100 nM) for 0, 4 and 6 h. ( f ) Densitometry revealed the relative levels of LDLR in bafilomycin A-treated cells ( n =3). ( g ) Representative images ( n =3) of LDLR on the surface of Wash1 f/f and Wash1 −/− MEFs determined by surface biotinylation assay. ( h ) Densitometry revealed relative LDLR surface levels ( n =3). ( i ) Wash1 f/f and Wash1 −/− MEFs were incubated with DiI-LDL for 30 min and imaged by fluorescence microscope. ( j ) Fluorescence intensity was quantified using ImageJ software and was normalized to the number of DAPI nuclei per image; >30 cells per condition were recorded. The results are presented as mean±s.e.m.; significance was calculated relative to the control group by unpaired Student's t -test; *** P <0.001.

Article Snippet: The following antibodies were used: rabbit polyclonal antibody against LDLR (PAB8804, Abnova, 1:200); mouse monoclonal antibody against COMMD1 (MAB7526, R&D Systems, 1:400); rabbit polyclonal antibody against EEA1 (2411, Cell Signaling Technologies, 1:250); goat polyclonal antibody against VPS35 (ab10099-100, Abcam, 1:1,000); rat monoclonal antibody against LAMP1 (553792, BD Biosciences, 1:500); rabbit polyclonal antibody against WASH1 (1:1,000); and rabbit polyclonal antibody against FAM21 (1:1,000) (ref. ).

Techniques: Immunofluorescence, Staining, Western Blot, Surface Biotinylation Assay, Incubation, Fluorescence, Microscopy, Software, Control

Journal: Nature Communications

Article Title: CCC- and WASH-mediated endosomal sorting of LDLR is required for normal clearance of circulating LDL

doi: 10.1038/ncomms10961

Figure Lengend Snippet: After internalization mediated by ARH, LDLR is sorted in endosomes . LDLR is directed to the lysosome for proteolysis—which is induced by either PCSK9 (refs , ) or IDOL —or retrieved and recycled back to the cell surface. SNX17 enhances LDL endocytosis likely by promoting LDLR retrieval , which might precede CCC and retromer/WASH-mediated endosomal trafficking. Through the interaction of the retromer component VPS35 with FAM21 the WASH complex and the CCC complex are recruited to the endosomes . Subsequently, CCC and WASH form a protein complex with LDLR; WASH facilitates the formation of branched actin patches to define restricted domains of the endosomes from where LDLR is sorted back to the cell surface. NDRG1 is required for the formation of MVB: loss of NDRG1 impairs LDL uptake and LDLR recycling, and causes the accumulation of IDOL-mediated ubiquitinated LDLR in MVB . Further research is needed to assess whether CCC and WASH are functionally related to SNX17, NDRG1 and to the lysosomal degradation pathways of LDLR. ARH, autosomal recessive hypercholesterolaemia protein; MVB, multivesicular body; PCSK9, proprotein convertase subtilisin/kexin type 9; Ub, ubiquitin; WASH, WASH1, FAM21, strumpellin, KIAA1033, CCDC53.

Article Snippet: The following antibodies were used: rabbit polyclonal antibody against LDLR (PAB8804, Abnova, 1:200); mouse monoclonal antibody against COMMD1 (MAB7526, R&D Systems, 1:400); rabbit polyclonal antibody against EEA1 (2411, Cell Signaling Technologies, 1:250); goat polyclonal antibody against VPS35 (ab10099-100, Abcam, 1:1,000); rat monoclonal antibody against LAMP1 (553792, BD Biosciences, 1:500); rabbit polyclonal antibody against WASH1 (1:1,000); and rabbit polyclonal antibody against FAM21 (1:1,000) (ref. ).

Techniques: Ubiquitin Proteomics

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Amyloid precursor protein (APP) traffics from the cell surface via endosomes for amyloid β (Aβ) production in the trans-Golgi network.

doi: 10.1073/pnas.1208635109

Figure Lengend Snippet: Fig. 4. VPS35 depletion delayed retrieval of APP to the TGN and Aβ pro- duction. For A–C, experiments were performed as in Fig. 1, and cells were transfected with empty vector (control) or two shRNA constructs directed to VPS35 or Rab9A (KD). (A) Immunoblot and (B) quantification of relative protein levels (mean ± SEM; n = 3), and (C) Aβ40 ELISA analysis (mean ± SEM; n ≥3; **P < 0.01 and ***P < 0.001, only P values < 0.05 shown). (D) Experiments were performed as in Fig. 2, and cells were transfected with pSUPER+GFP shRNA directed to VPS35 or Rab9A (KD). Representative cells with GFP signals are shown. (Scale bars: 10 μm.)

Article Snippet: Goat polyclonal anti-VPS35 antibody was obtained from Imgenex.

Techniques: Transfection, Plasmid Preparation, Control, shRNA, Construct, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Amyloid precursor protein (APP) traffics from the cell surface via endosomes for amyloid β (Aβ) production in the trans-Golgi network.

doi: 10.1073/pnas.1208635109

Figure Lengend Snippet: Fig. 5. Double knockdown of AP-4 μ and VPS35 retained APP in the early endosomes and reduced Aβ40 production. For A–C, experiments were per- formed as in Fig. 1, and cells were transfected with empty vector (control) or shRNA directed to AP-4 μ or pooled shRNA constructs (AP-4 μ and VPS35-1) directed to AP-4 μ and VPS35-1 (KD) for double knockdown. (A) Immunoblot. Antibody against the β-subunit of AP-4 was used because β- and μ-subunits of AP-4 reside in the same hemicomplex; depletion of AP-4 μ should mirror the loss of AP-4 β. (B) Quantification of relative protein levels (mean ± SEM; n = 3) (C) Aβ40 ELISA analysis (mean ± SEM; n = 3; *P < 0.05 and **P < 0.01). (D) Experiments were performed as in Fig. 2, and cells were transfected with pSUPER+GFP shRNA directed to AP-4 μ or pooled shRNA constructs (AP-4 μ and VPS35-1) directed to AP-4 μ and VPS35-1 (KD). Representative cells with GFP signals are shown. (Scale bars: 10 μm.)

Article Snippet: Goat polyclonal anti-VPS35 antibody was obtained from Imgenex.

Techniques: Knockdown, Transfection, Plasmid Preparation, Control, shRNA, Construct, Western Blot, Enzyme-linked Immunosorbent Assay