mab6228 rrid ab 3658562  (R&D Systems)

Journal: Nature Communications

Article Title: Control of lysosome function by the GTPase-activating protein TBC1D9B and its binding partner TMEM55B

doi: 10.1038/s41467-026-70345-y

Figure Lengend Snippet: a Immunoblot analysis of TBC1D9B -KO (upper panel) and TMEM55B -KO (lower panel) HeLa cells with antibodies against TBC1D9B and TMEM55B. GAPDH serves as a loading control. Two independent clones are shown. n = 3 independent experiments. b Lysosome dispersion in TBC1D9B -KO and TMEM55B -KO cells. Confocal microscopy images of TBC1D9B -KO and TMEM55B -KO cells stained for endogenous LAMP2 (green). Nuclei are stained with DAPI (blue). ns not significant; * = p < 0.05; ** = p < 0.01; wildtype vs. TBC1D9B KO p = 0.0303; wildtype vs. TMEM55B KO p = 0.0073. c Quantification of the number of peripheral lysosomes in wildtype, TBC1D9B -KO, and TMEM55B -KO cells, as determined by OrgaMapper. One-way ANOVA; ns not significant; * = p < 0.05; ** = p < 0.01. Mean ± SEM from n = 3 independent experiments. d Cartoon representation of the TMEM55B/TBC1D9B -KO effects on lysosomal positioning. The KO of TMEM55B or TBC1D9B leads to lysosome dispersion into the cell-periphery. e Quantification of the “percentage of lysosomes in the mobile fraction” of lysotracker red-stained wildtype, TMEM55B -KO, and TBC1D9B -KO cells determined by confocal live-cell imaging microscopy. One-way ANOVA; ns not significant; * = p < 0.05; wildtype vs. TBC1D9B KO p = 0.0225; wildtype vs. TMEM55B KO p = 0.0149; mean ± SEM from n = 1 experiment with wildtype n = 11, TBC1D9B KO n = 12, TMEM55B KO n = 11 measurements. Each measurement represents the mean of all tracks of a cell. f TBC1D9B-HA partially colocalizes with LAMP2 in TBC1D9B KO cells. Confocal images of TBC1D9B KO HeLa cells stained for TBC1D9B-HA (magenta) and endogenous LAMP2 (yellow). g Rescue of defective lysosome position in TBC1D9B -KO cells requires TBC1D9B GAP activity. Immunofluorescence microscopy for LAMP2 (green) of wildtype and untransfected TBC1D9B -KO cells or TBC1D9B -KO cells re-expressing wildtype TBC1D9B-HA (magenta) or a GAP-defective mutant (TBC1D9B RYQ/AAA -HA). Nuclei are stained with DAPI (blue). h Quantification of the number of peripheral lysosomes determined by OrgaMapper. One-way ANOVA; ns not significant; * = p < 0.05; vs. full length p = 0.0428. Mean ± SEM from n = 3 independent experiments.

Article Snippet: LAMP2 (clone H4B4) , DSHB , Cat# sc-18822 , RRID:AB_626858.

Techniques: Western Blot, Control, Clone Assay, Dispersion, Confocal Microscopy, Staining, Live Cell Imaging, Microscopy, Activity Assay, Immunofluorescence, Expressing, Mutagenesis

Journal: Nature Communications

Article Title: Control of lysosome function by the GTPase-activating protein TBC1D9B and its binding partner TMEM55B

doi: 10.1038/s41467-026-70345-y

Figure Lengend Snippet: a Confocal image of wildtype HeLa cells overexpressing TMEM55B (magenta) or TBC1D9B-HA (magenta) stained for endogenous LAMP2 (green). n = 3 independent experiments. b Cartoon representation of the effects of overexpression of TMEM55B or TBC1D9B on lysosome positioning. The overexpression of TMEM55B or TBC1D9B leads to perinuclear clustering of lysosomes. c Confocal images of wildtype HeLa cells overexpressing TBC1D9B RYQ/AAA- HA stained for HA (magenta) and endogenous LAMP2 (green). n = 3 independent experiments. d Pearson correlation coefficient of TBC1D9B/TBC1D9B RYQ/AAA -HA and LAMP2. Mean ± SD from pooled data of n = 3 independent experiments (TBC1D9B n 1 =, n 2 = 27, n 3 = 30; TBC1D9B-RYQ/AAA n 1 = 28, n 2 = 27, n 3 = 21). e Quantification of the number of peripheral lysosomes in each condition was determined by OrgaMapper. One-way ANOVA (left panel), t-test (right panel); ns not significant; * = p < 0.05; ** = p < 0.01. Mean ± SEM from n = 3 independent experiments; left panel: untransfected vs. full length p = 0.043; right panel; untransfected vs. transfected p = 0.0021.

Article Snippet: LAMP2 (clone H4B4) , DSHB , Cat# sc-18822 , RRID:AB_626858.

Techniques: Staining, Over Expression, Transfection

Journal: Nature Communications

Article Title: Control of lysosome function by the GTPase-activating protein TBC1D9B and its binding partner TMEM55B

doi: 10.1038/s41467-026-70345-y

Figure Lengend Snippet: a , b Loss of TBC1D9B or TMEM55B impairs starvation-induced perinuclear clustering of lysosomes. a Confocal images of fed and starved (6 h) wildtype, TBC1D9B -KO, and TMEM55B -KO HeLa cells stained for LAMP2. b Quantification of the number of peripheral lysosomes in the different genotypes and under the different conditions by OrgaMapper. One-way ANOVA; ns not significant; * = p < 0.05; ** = p < 0.01; *** = p < 0.001. Mean ± SEM from n = 4 independent experiments; wildtype fed vs. TBC1D9B KO fed p = 0.03; wildtype fed vs. TMEM55B KO fed p = 0.0013; wildtype starved vs. TBC1D9B KO starved p = 0.029; wildtype starved vs. TMEM55B KO starved p = 0.00047. c , d TBC1D9B and TMEM55B are required for the starvation-triggered induction of cathepsin D activity. c SiR-Lysosome staining of live fed- and starved cells (EBSS starved, 4 h) of the indicated genotypes. d Quantification of Cathepsin D activity in starved cells (relative to the unstarved/fed situation). One-way ANOVA; ns not significant; * = p < 0.05. Mean ± SEM from n = 3 independent experiments. e TBC1D9B depletion reduces autophagic flux in starved cells. (Left) immunoblotting and in-gel-fluorescence detection of total lysates of HeLa cells stably expressing Halo-LC3B upon treatment with the indicated siRNAs. (Right) quantification of Halo-LC3B cleavage. Halo band intensities were normalized by the sum of band intensities determined for Halo-LC3B and Halo. ( n = 3 independent experiments). Mean ± SEM from n = 3 independent experiments. Statistics: two-way ANOVA, followed by Tukey´s multiple comparisons test. (siControl-fed vs. siControl-starved: p = 0.002; siTBC1D9B-fed vs siTBC1D9B-starved: P = 0.2046; siControl-fed vs siTBC1D9B-fed: p = 0.9573; siControl-starved vs siTBC1D9B-starved: p = 0.0357).

Article Snippet: LAMP2 (clone H4B4) , DSHB , Cat# sc-18822 , RRID:AB_626858.

Techniques: Staining, Activity Assay, Western Blot, Fluorescence, Stable Transfection, Expressing

Journal: Nature Communications

Article Title: Control of lysosome function by the GTPase-activating protein TBC1D9B and its binding partner TMEM55B

doi: 10.1038/s41467-026-70345-y

Figure Lengend Snippet: a TurboID strategy for the identification of TBC1D9B-interacting proteins in iPSC-derived iNeurons. Created in BioRender. Damme, M. (2026) https://BioRender.com/nh1fa4i . b Untargeted label-free quantitation (LFQ) of the ARL8B proximity interactome in iNeurons. c TBC1D9B specifically associates with active ARL8B-GTP. Immunoprecipitation using GFP-Nanotrap antibodies from lysates of HeLa cells expressing different ARL8B variants (wildtype, Q75L, T34N) and TBC1D9B-HA. Samples were analyzed by immunoblotting for GFP and HA. GFP-transfected cells served as a negative control. n = 3 independent experiments. d In vitro pulldown experiment of recombinant purified TBC1D9B-eGFP incubated with different GST-ARL8B variants (Q75L, T34N) bound to beads. Recombinant GST served as a negative control. GST/GST-fusion proteins were visualized by Coomassie staining (false-color blue) and TBC1D9B-GFP by in-gel GFP fluorescence. Quantification of the relative binding of TBC1D9B-GFP to ARL8B Q75L/ T34N. One-sample t-test; two-sided; p = 0.0355. Mean ± SEM from n = 3 independent experiments. e In vitro pulldown experiment of recombinant purified GST-TMEM55B/GST alone and TBC1D9B-eGFP incubated with recombinant ARL8B lacking amino acids 1–17 (ARL8B delta17). GST-TMEM55B/GST were bound to beads. GST/GST-TMEM55B-GST-fusion protein was visualized by Coomassie staining (false-color blue), TBC1D9B-GFP, and ARL8B delta17 by immunoblot. n = 3 independent experiments. f Confocal images of wildtype HeLa cells overexpressing TBC1D9B-HA alone or together with the indicated eGFP-tagged-ARL8B variants (wildtype, Q75L, T34N; white) stained with antibodies against HA (magenta) and LAMP2 (green). Nuclei are stained with DAPI (blue). A representative experiment from 3 replicates is shown for the immunofluorescence panel. Fluorescence intensity profiles are depicted for the indicated cross-sections. The Pearson correlation coefficient of TBC1D9B-HA and LAMP2 is depicted. Bar graph: One-way ANOVA; mean ± SEM from n = 2 independent experiments; ns not significant; **** = p < 0.0001; TBC1D9B o/e vs. TBC1D9B + ARL8B Q75L o/e p = 2.48 × 10 −11 ; TBC1D9B + ARL8B o/e vs. TBC1D9B + ARL8B Q75L o/e p = 9.6 × 10 −13 ; TBC1D9B + ARL8B T34N o/e vs. TBC1D9B + ARL8B Q75L o/e p = 9.6 × 10 −13 ; TBC1D9B o/e n 1 = 29, n 2 = 32; TBC1D9B + ARL8B o/e n 1 = 47, n 2 = 35; TBC1D9B + ARL8B Q75L o/e n 1 = 51, n 2 = 31; TBC1D9B + ARL8B T34N o/e n 1 = 43, n 2 = 39.

Article Snippet: LAMP2 (clone H4B4) , DSHB , Cat# sc-18822 , RRID:AB_626858.

Techniques: Derivative Assay, Quantitation Assay, Immunoprecipitation, Expressing, Western Blot, Transfection, Negative Control, In Vitro, Recombinant, Purification, Incubation, Staining, Fluorescence, Binding Assay, Immunofluorescence

Journal: Nature Communications

Article Title: Control of lysosome function by the GTPase-activating protein TBC1D9B and its binding partner TMEM55B

doi: 10.1038/s41467-026-70345-y

Figure Lengend Snippet: a Alphafold3 model for TBC1D9B (orange) bound to ARL8B (ruby-colored) in the presence of GTP (yellow sticks). The residues mutated in the TBC1D9B RYQ/AAA version are shown as spheres and highlighted in red. b Closeup view of theTBC1D9B-(GTP)-ARL8B interface. c GTPase activity of 6 μM GTP-bound GST-ARL8B was measured using the GTPase-Glo assay in the presence of 0.5 μM TBC1D9B or TBC1D9B RYQ/AAA at different incubation times. GTPase-Glo™ detects the remaining GTP upon the conversion to ATP; ATP is quantified by the addition of detection reagent and emitted luminescence. The change of luminescence indicates the amount of hydrolyzed GTP in the reaction. Data are presented as mean values ± SEM, n = 3 independent experiments. Two-way ANOVA, Bonferroni’s multiple comparisons test; GST-ARL8B + TBC1D9B vs. GST-ARL8B, at 30 min, p = 1.005 × 10 −5 ; at 60 min, p < 1.4 × 10 −13 ; at 90 min, p < 1 × 10 −14 . d Confocal images of wildtype, TMEM55B knockout, and TBC1D9B knockout HeLa cells stained for LAMP2 and ARL8B-GFP. Left, Pearson correlation coefficient between LAMP2 and ARL8B. One-way ANOVA; mean ± SEM from n = 1 experiment with WT n = 41, TBC1D9B KO n = 39, TMEM55B KO n = 31; ns not significant; **** = p < 0.0001; Wildtype vs. TBC1D9B KO p = 2.37 × 10 −6 ; Wildtype vs. TMEM55B KO p = 5.47 × 10 −7 .

Article Snippet: LAMP2 (clone H4B4) , DSHB , Cat# sc-18822 , RRID:AB_626858.

Techniques: Activity Assay, Glo Assay, Incubation, Knock-Out, Staining

Journal: Nature Communications

Article Title: Control of lysosome function by the GTPase-activating protein TBC1D9B and its binding partner TMEM55B

doi: 10.1038/s41467-026-70345-y

Figure Lengend Snippet: a Confocal images of wildtype, TMEM55B -KO, and TBC1D9B -KO HeLa cells treated with scrambled siRNA or siRNA against ARL8B and stained for endogenous LAMP2. b The fraction of cells with peripheral lysosomes was quantified using OrgaMapper. One-way ANOVA (Šídák’s multiple comparisons test), ** p < 0.01, *** p < 0.001. wildtype scr vs. wildtype siARL8B p = 0.1093; wildtype scr vs. TBC1D9B KO p = 0.0047; scr wildtype scr vs. TMEM55B KO p = 0.0026; scr TBC1D9B KO scr vs. TBC1D9B KO p = 0.0031; siARL8B TMEM55B KO scr vs. TMEM55B KO siARL8B p = 0.0003; mean ± SEM from n = 3 independent experiments. c Confocal images of wildtype and ARL8A/B double-KO (DKO) cells treated with scrambled siRNA or siRNA against TBC1D9B and stained for LC3. d LC3-signal intensity was quantified and expressed as a fold change of scrambled-treated wildtype cells. One-way ANOVA, ns = not significant, **** p < 0.0001. Mean ± SEM from n = 3 independent experiments. e Schematic representation of the proposed model: TMEM55B binds TBC1D9B, which in turn acts as a GAP for ARL8B to facilitate nucleotide exchange and, thereby, membrane association and effector recruitment. TMEM55B-association putatively recruits TBC1D9B to the lysosomal surface and/ or mediates a conformational change to activate TBC1D9B.

Article Snippet: LAMP2 (clone H4B4) , DSHB , Cat# sc-18822 , RRID:AB_626858.

Techniques: Staining, Membrane

Journal: bioRxiv

Article Title: Loss of Lamp2a-dependent chaperone-mediated autophagy drives dry AMD-like retinal pathology in mice and is rescued by BK channel activation

doi: 10.64898/2026.03.19.712761

Figure Lengend Snippet: (A) Cartoon representation of Lamp2a knockout mouse generation. (B) Confocal microscopy imaging validating deletion of Lamp2a. Red staining reflects Lamp2a, green staining shows Lamp2b. Scale bar =300 µm (C) Immunoblot of RPE/choroid lysate showing loss of Lamp2a expression in Lamp2a -/- mice compared to wild type (WT) age matched mice. (D) FAF images showing high-intensity autofluorescence spots in WT and Lamp2a -/- mice. Lamp2a deficiency results in an age-dependent increase in high-intensity autofluorescence spots compared with WT mice. (E) Quantification of high-intensity autofluorescence spots (n=8). Statistical analysis was performed using Two-way ANOVA with Dunnett’s multiple comparisons test. ***P < 0.001; ****P < 0.0001 . (F) Retinal B-scans of Lamp2a -/- and WT mice. Significant structural disturbance in retinal layers was seen in 12 months Lamp2a -/- mice as compared to WT aged mice and Lamp2a -/- younger mice (n=6). Scale bar =100 µm (width) and 60 = µm (height). Values are expressed as mean ± SD. (G) Representative scotopic ERG response of 6-7-month-old WT and Lamp2a -/- mice. (H) b-wave quantification indicating decline in scotopic response in Lamp2a -/- mice relative to age-matched wild type (n=6 mice/group). (I) Representative scotopic response of 10-12-month-old WT and Lamp2a -/- mice. (J) B-wave quantification shows significant decline in scotopic response in Lamp2a -/- mice relative to age-matched WT (n=8 mice/group). (K) Representative maximal response of 6-7-month-old WT and Lamp2a -/- mice. (L) b-wave amplitude quantification shows decreased scotopic response in 6-7-month-old Lamp2a -/- mice relative to age-matched WT (n=6 mice/group). (M) Representative maximal response of 10-12 months old WT and Lamp2a -/- mice. (N) b-wave amplitude quantification shows significant decrease in scotopic response in 10-12-month-old Lamp2a -/- mice relative to age-matched wild type (n=8 mice/group). (O) Representative photopic response of 6-7-month-old WT and Lamp2a -/- mice. (P) b-wave amplitude quantification shows photopic response changes in 6-7-month-old Lamp2a -/- mice relative to age-matched WT (n=6 mice/group). (Q) Representative photopic response of 10-12-month-old WT and Lamp2a -/- mice. (R) b-wave amplitude quantification shows pronounced decrease in photopic response in 10-12-month-old Lamp2a -/- mice relative to age-matched WT (n=8 mice/group). Statistical differences were analyzed using an unpaired two-tailed student’s t-test. Level of significance was considered at p value < 0.05 .

Article Snippet: Adult male Lamp2a knockout mice ( Lamp2a y/- ), female heterozygous Lamp2a +/- and their wild type (WT) littermates ( Lamp2a y/+ ) were obtained from Cyagen (Serial Number KOCMP-16784-Lamp2-B6J-VA; strain C57BL/6J-Lamp2 em1Cya ).

Techniques: Knock-Out, Confocal Microscopy, Imaging, Staining, Western Blot, Expressing, Two Tailed Test