MA1-066

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescence analysis of Mannose 6 Phosphate Receptor/CD222 was done on 70% confluent log phase HepG2 cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 1% BSA for 1 hour at room temperature. The cells were labeled with Mannose 6 Phosphate Receptor/CD222 (2G11) Mouse Monoclonal Antibody (Product # MA1-066) at 2 µg/mL in 0.1% BSA and incubated for 3 hours at room temperature and then labeled with Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A28175) at a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). F-actin (Panel c: red) was stained with Alexa Fluor® 555 Rhodamine Phalloidin (Product # R415, 1:300). Panel d is a merged image showing cytoplasmic localization. Panel e is a no primary antibody control. The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescent analysis of Mannose 6-Phosphate Receptor using Anti-Mannose 6-Phosphate Receptor Monoclonal Antibody (2G11) (Product # MA1-066) shows staining in Hela Cells. Mannose 6-Phosphate Receptor staining (green), F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control) or with or an antibody recognizing Mannose 6-Phosphate Receptor (Product # MA1-066) at a dilution of 1:20 over night at 4°C, washed with PBS and incubated with a DyLight-488 conjugated secondary antibody (Product # 35503, Goat Anti-Mouse). Images were taken at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescent analysis of Mannose 6-Phosphate Receptor using Anti-Mannose 6-Phosphate Receptor Monoclonal Antibody (2G11) (Product # MA1-066) shows staining in HepG2 Cells. Mannose 6-Phosphate Receptor staining (green), F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control) or with or an antibody recognizing Mannose 6-Phosphate Receptor (Product # MA1-066) at a dilution of 1:20 over night at 4°C, washed with PBS and incubated with a DyLight-488 conjugated secondary antibody (Product # 35503, Goat Anti-Mouse). Images were taken at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescent analysis of Mannose 6-Phosphate Receptor using anti-Mannose 6-Phosphate Receptor monoclonal antibody (Product # MA1-066) shows staining in HMVEC Cells.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescence analysis of IGF2R was performed using 70% confluent log phase Hep G2 cells. The cells were fixed with 4% paraformaldehyde for 5 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 2% BSA for 45 minutes at room temperature. The cells were labeled with IGF2R Monoclonal Antibody (2G11) (Product # MA1-066) at 5 µg/mL concentration in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Donkey anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32766) (1:2000 dilution), for 45 minutes at room temperature (Panel a: Green). Nuclei (Panel b: Blue) were stained with ProLong™ Diamond Antifade Mountant with DAPI (Product # P36962). F-actin (Panel c: Red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing Golgi and Cytoplasmic localization. Panel e represents low expressing cell line A549. Panel f represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 3. CI-MPR segregates in SNX1/2-SNX5/6 tubular profiles which are not decorated with retromer. (A) Endogenous CI-MPR localizes to a highly packed vesicular cluster that colocalized with the TGN and partially colocalized with retromer-positive endosomes. HeLa cells were fixed and immunostained for endogenous TGN46 (TGN marker), VPS35 (retromer), SNX1, and SNX2 (both retromer-linked SNX-BARs). Bars: (main images) 20 um; (zooms) 5 um. (B, top) CI-MPR colocalizes with SNX2-positive subdomains on enlarged endosomes. HeLa cells were transfected with BFP-Rab5Q79L and immunostained for endogenous CI-MPR, SNX2, and VPS35 after 48 h. The dashed line in the top left image indicates the contour of the nucleus, and the dashed line on the top right refers to the enlarged endosome from which the intensity line scan was measured. Bars: (main images) 10 um; (zooms) 5 um. (B, bottom) Line scan of signal intensity across the circumference of enlarged endosome. (B, right) Distribution of CI-MPR in retromer subdomains; n = 3 independent experiments. CI-MPR signal was quantified in 36 enlarged endosomes (means +- SEM). (C) Overexpression of a WT GFP-CI-MPR chimera leads to the formation of extended CI-MPR tubules, some of which are positive for endogenous SNX1. HeLa cells were transfected with GFP-CI-MPR chimera WT and immunostained for SNX1 after 48 h. Bars: (main images) 20 um; (zooms) 10 um. (D) A subpopulation of GFP-CI-MPR chimera tubules is decorated with endogenous SNX1 but not

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 7. Gene editing confirms the essential role of SNX1/2-SNX5/6 in endosome-to-TGN recycling of CI-MPR. (A, left) CI-MPR levels and CI-MPR steady-state localization in retromer and SNX1/2-SNX5/6 KO cells. HeLa cells were transfected with CRISPR-Cas9 plasmids against SNX1 and SNX2, SNX5 and SNX6, or VPS35. 96 h after transfection, endogenous protein levels were analyzed by Western blotting, and cells were fixed and stained for endogenous CI-MPR. n = 3 independent experiments (one-way ANOVA compared with parental HeLa). Molecular masses are given in kilodaltons. (A, middle) Representative images. Bars, 20 um. (A, right) quantification of the percentage of cells displaying each phenotype. n = 3 blindly scored independent experiments; parental, 116 cells; VPS35, 106 cells; SNX5 + SNX6, 111 cells; VPS35, 106 cells. (B) Immunofluorescence of endogenous CI-MPR and endogenous SNX1 and VPS35 in retromer KO cells and SNX1 and SNX2 KO cells. Bars: (top) 20 um; (bottom) 5 um. (C) Immunofluorescence and colocalization analysis of endogenous CI-MPR and TGN marker TGN46 and early endosomal marker EEA1 in SNX1 and SNX2, SNX5 and SNX6, and VPS35 CRISPR-Cas9 KO HeLa cells. Bars: (main images) 20 um; (insets) 10 um. (C, top right) n = 3 independent experiments; parental HeLa, 80 cells; SNX1+2 KO, 99 cells; SNX5+6, 81 cells; VPS35 KO, 76 cells. (C, bottom right) n = 4 independent experiments; parental HeLa, 102 cells, SNX1+2 KO, 114 cells; SNX5+6, 104 cells; VPS35 KO, 102 cells (means +- SEM

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 8. Clonal HeLa KO cell lines recapitulate the SNX1/2-SNX5/6 mediated retromer-independent retrograde transport of CI-MPR. (A) Clonal cell lines were isolated from a heterogeneous population of CRISPR-Cas9 KO. Two independent lines were biochemically characterized as SNX1+2 KOs, two as SNX5+6 KOs, two as VPS35 KOs, and three as parental clonal lines. Molecular masses are given in kilodaltons. (A, right) CI-MPR levels were analyzed by Western blotting. n = 3 independent experiments. (B, left) Immunofluorescence and colocalization analysis of endogenous CI-MPR, TGN marker TGN46, and early endosomal marker EEA1 in parental HeLa and clonally selected KO lines. Bars, 20 um. (B, top right two graphs) n = 3 independent experiments; parental HeLa, 58 cells; parental c8, 79 cells; parental c21, 81 cells; SNX1+2 KO c4, 75 cells; SNX1+2 KO c16, 70 cells; SNX5+6 KO c13, 82 cells; SNX5+6 KO c18, 89 cells; VPS35 KO c5, 83 cells; VPS35 KO c7, 72 cells. (B, bottom right two graphs) n = 3 independent experiments; parental HeLa, 67 cells; parental c8, 72 cells; parental c21, 87 cells; SNX1+2 KO c4, 83 cells; SNX1+2 KO c16, 80 cells; SNX5+6 KO c13, 84 cells; SNX5+6 KO c18, 98 cells; VPS35 KO c5, 73 cells; VPS35 KO c7, 68 cells (means +- SEM; one-way ANOVA compared with parental HeLa. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.001).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 6 Colocalization of MLG with markers for recycling endosomes. A synchronized fusion assay was performed as described for Fig. 4B , the cells were fixed at t-60, stained for (A) LAMP-1, (B) mannose-6-phosphate receptor, (C) Tfn-TR, or (D) Rab11 and then examined by LSCM (Bars = 10 um). Percent colocalization of MLG with the indicated marker from approximately 20 individual cells from two independent experiments is shown in the upper right-hand corner of the merged images.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 3 Colocalization of M6PR with Rab7-, LAMP1-, and Rab7/LAMP1-vesicles. ( A ) Confocal microscopy image of ECFP-Rab7 (blue), LAMP1-EYFP (green), and an antibody against M6PR labeled with a Cy5-labeled secondary antibody (red). Smaller images show a M6PR-positive (red) Rab7-vesicle (blue, top) and a M6PR-negative LAMP1-vesicle (green, bottom). ( B ) A large fraction of Rab7- and Rab7/LAMP1-vesicles are positive for M6PR; 57+-7% and 63+-15%, respectively. A smaller fraction of LAMP1-vesicles are positive for M6PR, 23+-10%. Error bars show standard deviations. P-values0.05. The graph shows the analysis of 10-20 of each type of vesicle per cell in 10 cells from 4 distinct experiments. Similar results were obtained for BS-C-1 cells stably expressing ECFP-Rab7 and for HeLa cells ( Figure S4 ). Unmerged images are shown in Figure S5 .

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 5. The COR1C CC limits bud actin to facilitate ER contact, endosome fission, and CI-M6PR sorting. (A) Representative images of LE buds stable for duration of acquisition in conditions that did not rescued fission rate (C). COS-7 cells were cotransfected with COR1A/1B/1C siRNAs to deplete all type I coronins and with GFP-Rab7 (LE, magenta), mCh-FAM21 (WASH complex, green), and with either Halo E-vec, siRES COR1C DeltaCC-Halo, or siRES COR1C ACT- DeltaCC-Halo. Arrows indicate bud of interest. (B) Representative images of LE fission events in conditions that rescued fission rate (C). COS-7 cells were cotransfected with COR1A/1B/1C siRNAs to deplete all type I coronins and with GFP-Rab7 (LE, magenta), mCh-FAM21 (WASH complex, green), and with either siRES COR1C-Halo, siRES COR1C ACT--Halo, or siRES COR1C CC-Halo. Arrows indicate bud of interest. (C) Quantification of data in A and B. Graph shows percentage of FAM21-labeled LE buds that underwent fission per cell during a 2-min time lapse. Note that only constructs containing the CC were able to restore fission. Data for graph from Halo E-vec: 139 endosomes in n = 15 cells; siRES COR1C: 122 endosomes in n = 15 cells; siRES COR1C ACT-: 213 endosomes in n = 17 cells; siRES COR1C DeltaCC: 281 endosomes in n = 18 cells; siRES COR1C ACT- DeltaCC: 296 endosomes n = 17 cells; and siRES COR1C CC: 331 endosomes in n = 22 cells, performed in triplicate. (D) Representative images of the M6PR trafficking assay. The relative fluorescenc

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig. 4 Bevacizumab is somewhat co-localized with CI-M6PR. After treatment of iBREC with the humanized IgG bevacizumab for 4 h, cells were immunostained to visualize the IgG (red) or CI-M6PR (green). The prominent CI-M6PR-specific signals close to the nucleus only to a small extent overlapped with those of bevacizumab (yellow arrowheads). Scale bar, 10 mum