beta adaptin  (Santa Cruz Biotechnology)

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: RAB23 facilitates clathrin-coated nascent vesicle formation at the plasma membrane and modulates cell signaling

doi: 10.1007/s00018-025-05694-w

Figure Lengend Snippet: HA-RAB23 co-localizes with early and late endosomal markers and interacts with the β-adaptin subunit of the clathrin adaptor protein 2 (AP-2) complex. a, b Co-localization analysis ( a ) of HA-RAB23 with endocytic pathway-specific vesicle markers EEA1, RAB5, RAB7, RAB11 and with autophagy marker LC3 A/B in MG-63 cells. Images show HA-RAB23 (red) co-localizes with the early endosomal markers EEA1 and RAB5 (green) in different locations in the cell including in the cell periphery (inset). HA-RAB23 shows robust co-localizations with late endosomal marker RAB7 (green) and autophagy marker LC3 A/B (green). HA-RAB23 shows little or no co-localization with recycling endosomal marker RAB11. Scale bar: 10 µm. Quantification ( b ) of HA-RAB23 co-localizations with EEA1, RAB5, RAB7, LC3 A/B and RAB11 using Pearson’s correlation coefficient r = 0–0.19 (very low co-localization), r = 0.2–0.39 (low co-localization), r = 0.4–0.59 (moderate correlation), r = 0.6–0.79 (high correlation) and r = 0.8–1.0 (very high correlation). n = 3 (total number of cells 25–30). c Co-localization analysis of HA-RAB23 with endocytic route-specific markers clathrin, caveolin 1 and β-adaptin subunit of the clathrin adaptor protein 2 (AP-2) complex in MG-63 cells. Images show robust co-localization of HA-RAB23 (red) with clathrin (green) and β-adaptin (green), HA-RAB23 shows low co-localization with caveolin 1 (green). Scale bar: 10 µm. n = 3 (total number of cells 20–25). d Protein co-immunoprecipitation using IgG and anti-HA antibody on un-transfected and transfected MG-63 cells, respectively. Co-immunoprecipitation followed by western blotting using anti-β adaptin antibody detected the β-adaptin band in the anti-HA co-immunoprecipitated sample at the same molecular weight (105 kDa) to that of input β-adaptin protein observed in transfected and un-transfected cells. Western blotting using anti-RAB23 antibody detected HA-RAB23 protein in the anti-HA co-immunoprecipitated sample at 30 kDa. Western blotting using anti-β actin antibody detected β-actin protein in the un-transfected and transfected inputs at 42 kDa (n = 4 independent blots). PM; plasma membrane. e Co-localization of GFP-RAB23 with AP-2 subunit α-adaptin 1 and α-adaptin 2 in MG-63 cells transfected with RAB23-pEGFP-C1 expression vector. Images show that GFP-RAB23 co-localizes with α-adaptin 1 in the cell periphery. GFP-RAB23 shows little or no co-localizations with α-adaptin 2. Nuclear staining (blue). n = 3 (total number of cells ⁓20). Scale bar, 10 µm. f Protein co-immunoprecipitation using IgG and anti-HA antibody on un-transfected and transfected (HA-RAB23 pcDNA3.1 expression plasmid) MG-63 cells, followed by western blotting using anti-α-adaptin 1 antibody failed to detect α-adaptin 1 protein band (105 kDa) in the co-immunoprecipitated sample. Western blotting using anti-RAB23 antibody detected HA-RAB23 protein in the anti-HA co-immunoprecipitated sample at 30 kDa. Western blotting using anti-β actin antibody detected β-actin protein in the un-transfected and transfected inputs at 42 kDa. (n = 2 independent blots). g Protein co-immunoprecipitation using IgG and anti-HA antibody on un-transfected and transfected (HA-RAB23 pcDNA3.1 expression plasmid) MG-63 cells, followed by western blotting using anti-α-adaptin 2 antibody failed to detect α-adaptin 2 protein band (105 kDa) in co-immunoprecipitated sample. Western blotting using anti-RAB23 antibody detected HA-RAB23 protein in the anti-HA co-immunoprecipitated sample at 30 kDa. Western blotting using anti-β actin antibody detected β-actin protein in the un-transfected and transfected inputs at 42 kDa. (n = 2 independent blots). h Model suggests RAB23, clathrin and AP-2 might function at the cell membrane

Article Snippet: Anti-β adaptin antibody-bound protein samples were eluted in 2x samples buffer boiled at 95 °C for 6 minutes and analyzed by 4–20% SDS–PAGE (Biorad; 456–1094) and western blotting with rabbit primary anti-β-adaptin, anti-α adaptin 1, anti-α adaptin 2, anti-clathrin, anti-RAB23 and anti-β actin antibodies.

Techniques: Marker, Immunoprecipitation, Transfection, Western Blot, Molecular Weight, Clinical Proteomics, Membrane, Expressing, Plasmid Preparation, Staining

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: RAB23 facilitates clathrin-coated nascent vesicle formation at the plasma membrane and modulates cell signaling

doi: 10.1007/s00018-025-05694-w

Figure Lengend Snippet: RAB23, clathrin and AP-2 co-localize during nascent vesicle formation. a Triple co-localization of GFP-RAB23 with clathrin and β-adaptin subunit of the clathrin adaptor protein 2 (AP-2) in MG-63 cells transfected with RAB23-pEGFP-C1 expression vector. Images show RAB23 (green) co-localizes together with clathrin (red) and the β-adaptin (magenta) and show triple co-localization (arrow, inset) in the cell periphery. n = 3 (total number of cells 12–15). Scale bar, 10 µm. b Control co-localization of GFP with clathrin and β-adaptin subunit of the clathrin adaptor protein 2 (AP-2) in MG-63 cells transfected with EGFP-pcDNA3.1 expression vector. GFP (green) showed no co-localization with clathrin (red) and the β-adaptin (magenta). n = 3 (total number of cells 12–15). Scale bar, 10 µm

Article Snippet: Anti-β adaptin antibody-bound protein samples were eluted in 2x samples buffer boiled at 95 °C for 6 minutes and analyzed by 4–20% SDS–PAGE (Biorad; 456–1094) and western blotting with rabbit primary anti-β-adaptin, anti-α adaptin 1, anti-α adaptin 2, anti-clathrin, anti-RAB23 and anti-β actin antibodies.

Techniques: Transfection, Expressing, Plasmid Preparation, Control

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: RAB23 facilitates clathrin-coated nascent vesicle formation at the plasma membrane and modulates cell signaling

doi: 10.1007/s00018-025-05694-w

Figure Lengend Snippet: RAB23 deficiency changes the pattern of transferrin distribution and reduces transferrin uptake. a Time-lapse imaging of transferrin internalization dynamics in the presence of membrane dye (CellBrite green) in WT ( Linked with Video ) and Rab23 -/- (Linked with Video ) cells. Cells were starved for 1 hour in growth medium containing 0.1% FBS. Initially, membrane was stained with membrane dye (green) followed by 5 minutes transferrin (red) pulse. Cells were then washed, and time-lapse imaging was performed for 5 minutes (frame rate 1-s interval) with sequential dual laser excitation at 594 nm and 488 nm. The first time frame indicates the starting time (T = 5 min) of the time-lapse, the mid-time frame indicates when the time reaches 7.5 minutes and the last time frame indicates the end time (T = 10 min) of the time-lapse. WT cells show robust internalization of transferrin (arrow, Video ) while Rab23 -/- cells retain transferrin at the cell membrane (Video ), Or, after being initial internalization of transferrin, many transferrin patches repulse from cytoplasm to the periphery of the cell (arrow, last time frame). The dash line indicates the boundary of the cell. 10–12 cells in each group (n = 3 independent experiments). Scale bar, 10 µm. b, c Western blotting ( b ) and subsequent quantifications ( c ) show uptake of transferrin by cultured WT and Rab23 -/- mouse calvaria-derived primary cells at 5, 10, 30, 45, 60 and 120 minutes. Cells were starved and allowed to uptake transferrin with 0.1% FBS. α-Tubulin was used for normalizing the transferrin level. n = 3 independent experiments. Data represented as mean ± SD, paired Student’s t –test was used. Statistical significance was defined as a P ˂ 0.05 (*), ˂ 0.02 (**) and ˂ 0.005 (***). d–f Time-lapse imaging of transferrin (red dots in black background) accumulation pattern in the periphery of WT (Linked with Video ) and Rab23 -/- (Linked with Video ) cells. Cells were starved for 1 hour in growth medium containing 0.1% FBS followed by 5 minutes transferrin pulse. Cells were then washed, and time-lapse imaging was performed for 5 minutes (frame rate 1-s interval) with laser excitation at 594 nm. The first time frame indicates the starting time (T = 5 min) of the time-lapse and the last time frame indicates the end time (10 min) of the time-lapse. Segmented cell periphery shows transferrin (red dots, T = 5 and 10 mins). In WT cells, transferrin internalized (white and green arrows in their corresponding i, ii, i´ and ii´insets) efficiently from the cell periphery and the intensity of transferrin reduced after 5 minutes (T = 10, dotted rectangles) while Rab23 -/- cells transferrin internalized (white and green arrows in their corresponding i, ii, i´ and ii´insets) inefficiently from the cell periphery and transferrin retain at the cell periphery at this time (T = 10, dotted rectangles) ( d ). Scale bar: 20 µm. A model image represents the cell periphery (CellBrite green) in the segmented cell ( e ). Quantification of transferrin uptake in the cell periphery that showed in WT and Rab23 -/- cells by time-lapse imaging. Cells were initially segmented to define the cell periphery and quantified the intensity of transferrin in the first frame (T = 5) and last frame (T = 10 minutes) of time-lapse. Rab23 -/- cells retain more transferrin at the cell periphery compared to WT cells (f). 10–12 cells in each group (n = 3 independent experiments). Data represented as mean ± SD, paired Student’s t –test was used. Statistical significance was defined as a P ˂ 0.05 (*), ˂ 0.02 (**). g, h Co-localization ( g ) analysis of transferrin (red) and AP-2 (β-adaptin, green) at 5 and 10 minutes in WT and Rab23 -/- primary cells. Cells were starved and allowed to uptake transferrin with 0.1% FBS. WT cells show more transferrin and AP-2 positive patches at the cell periphery at 5 mins (upper inset, arrow) and many patches already internalized at this time (lower inset), while Rab23 -/- cells show less co-localized transferrin with AP-2 at the cell periphery (upper inset). At 10 minutes Rab23 -/- cells show more co-localization of transferrin with AP-2 (yellow, arrow) compared to WT cells. n = 3 independent experiments (total number of cells 25–30). Scale bar, 10 µm. Quantification ( h ) of transferrin co-localizations with AP-2 at 5- and 10 minutes using Pearson’s correlation coefficient r = 0–0.19 (very low co-localization), r = 0.2–0.39 (low co-localization), r = 0.4–0.59 (moderate correlation)

Article Snippet: Anti-β adaptin antibody-bound protein samples were eluted in 2x samples buffer boiled at 95 °C for 6 minutes and analyzed by 4–20% SDS–PAGE (Biorad; 456–1094) and western blotting with rabbit primary anti-β-adaptin, anti-α adaptin 1, anti-α adaptin 2, anti-clathrin, anti-RAB23 and anti-β actin antibodies.

Techniques: Imaging, Membrane, Staining, Western Blot, Cell Culture, Derivative Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: RAB23 facilitates clathrin-coated nascent vesicle formation at the plasma membrane and modulates cell signaling

doi: 10.1007/s00018-025-05694-w

Figure Lengend Snippet: RAB23 facilitates the assembly of clathrin-coat to the adaptor protein AP-2 (β-adaptin). a, b Dynamics of co-localization between PICALM and clathrin, AP-2 and clathrin for 5 and 10 minutes in WT and Rab23 -/- cells. Cells were initially starved followed by stimulated with the osteogenic medium. At 5 minutes (A) PICALM and clathrin, AP-2 and clathrin showed less co-localization coefficient in Rab23 -/- cells compared to WT cells ( a ). n = 3 independent experiments (total number of cells ≈ 30). At 10 minutes ( b ), AP-2 and clathrin showed significantly less co-localization coefficient in Rab23 -/- cells when compared to WT cells. n = 3 independent experiments (total number of cells ≈ 30). Scale bar, 10 µm. Data represented as mean ± SD, paired Student’s t –test was used. Statistical significance was defined as a P ˂ 0.05 (*). c A model delineating clathrin-mediated vesicle biogenesis at the plasma membrane upon binding with ligand (red) to receptor (i, green). Vesicle biogenesis starts with the recruitment of adaptor protein-2 (AP-2, sky blue). AP-2 interacts with ligand-receptor or cargoes and forms a prebudding structure (ii). Clathrin assembly protein PICALM (black) recruits clathrin (yellow) and forms a cage-like vesicle coat layer around the AP-2 mediated prebudding layer (iii). RAB23 modulates multiple steps: assembly of PICALM and AP-2 (blue), assembly of PICALM and clathrin (blue) and assembly of AP-2 and clathrin (blue) during nascent vesicle formation

Article Snippet: Anti-β adaptin antibody-bound protein samples were eluted in 2x samples buffer boiled at 95 °C for 6 minutes and analyzed by 4–20% SDS–PAGE (Biorad; 456–1094) and western blotting with rabbit primary anti-β-adaptin, anti-α adaptin 1, anti-α adaptin 2, anti-clathrin, anti-RAB23 and anti-β actin antibodies.

Techniques: Clinical Proteomics, Membrane, Binding Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: RAB23 facilitates clathrin-coated nascent vesicle formation at the plasma membrane and modulates cell signaling

doi: 10.1007/s00018-025-05694-w

Figure Lengend Snippet: RAB23 deficiency causes aberrant vesicle formation and alters BMP2 signaling. a, b Co-localization ( a ) analysis of clathrin (red) and AP-2 (β-adaptin, green) upon unstimulated and BMP2 stimulated WT and Rab23 -/- primary cells. Cells were starved and stimulated with BMP2 containing medium supplemented with 0.1% FBS for 5 and 10 minutes. BMP2 unstimulated cells received only fresh growth medium containing 0.1% FBS. Both WT and Rab23 -/- unstimulated primary cells showed co-localization of clathrin and AP-2, however, upon BMP2 stimulation for 5 minutes WT cells showed robust formation of the vesicle (i, ii) and vesicle like structure in the cell periphery, Rab23 -/- cells lacked the robustness and showed aberrant vesicle like structure (i´, ii´). At 10 minutes of BMP2 stimulation, WT cells showed a reduced number of vesicles and vesicle-like structures compared to 5 minutes of BMP2 stimulation in WT cells, whereas Rab23 -/- cells showed a drastic reduction of such structures at this time point. Scale bar, 20 µm. Quantification ( b ) of vesicle and vesicle-like structure in WT and Rab23 -/- primary cells without and with BMP2 stimulation for 5 and 10 minutes. (n = 3) (total number of cells ≈ 45). White arrowhead indicates vesicle-like structure. Data represented as mean ± SD, paired Student’s t –test was used. Statistical significance was defined as a P ˂ 0.05 (*), ˂ 0.02 (**) and ˂ 0.005 (***). c, d Protein co-immunoprecipitation ( c ) using IgG and AP-2 (β-adaptin) antibody on samples obtained from mouse WT and Rab23 -/- calvaria derived primary cells. Before co-immunoprecipitation cells were serum starved for 1 hour followed by BMP2 was added at a concentration of 75 ng/ml in the culture medium and kept at 37 °C for 5 minutes. Western blotting on co-immunoprecipitated samples using anti-β-adaptin and anti-clathrin antibodies detected the β-adaptin (105 kDa) band and clathrin (190 kDa) band at the same molecular weight as that of the input β-adaptin and input clathrin protein in WT and Rab23 -/- samples but not in the control IgG immunoprecipitated sample. Western blotting using anti-β actin antibody detected β-actin protein in the WT and Rab23 -/- inputs at 42 kDa. Quantification ( d ) of interaction (clathrin/β-adaptin subunit of AP-2) (n = 3 independent blots). Data represented as mean ± SD, paired Student’s t –test was used. Statistical significance was defined as a P ˂ 0.05 (*)

Article Snippet: Anti-β adaptin antibody-bound protein samples were eluted in 2x samples buffer boiled at 95 °C for 6 minutes and analyzed by 4–20% SDS–PAGE (Biorad; 456–1094) and western blotting with rabbit primary anti-β-adaptin, anti-α adaptin 1, anti-α adaptin 2, anti-clathrin, anti-RAB23 and anti-β actin antibodies.

Techniques: Immunoprecipitation, Derivative Assay, Concentration Assay, Western Blot, Molecular Weight, Control

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: RAB23 facilitates clathrin-coated nascent vesicle formation at the plasma membrane and modulates cell signaling

doi: 10.1007/s00018-025-05694-w

Figure Lengend Snippet:

Article Snippet: Anti-β adaptin antibody-bound protein samples were eluted in 2x samples buffer boiled at 95 °C for 6 minutes and analyzed by 4–20% SDS–PAGE (Biorad; 456–1094) and western blotting with rabbit primary anti-β-adaptin, anti-α adaptin 1, anti-α adaptin 2, anti-clathrin, anti-RAB23 and anti-β actin antibodies.

Techniques: Marker, Recombinant, Lysis, Blocking Assay, Protease Inhibitor, Transfection, Bicinchoninic Acid Protein Assay, Derivative Assay, Plasmid Preparation, Software, Imaging

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: RAB23 facilitates clathrin-coated nascent vesicle formation at the plasma membrane and modulates cell signaling

doi: 10.1007/s00018-025-05694-w

Figure Lengend Snippet:

Article Snippet: Rabbit anti-AP2B1 (β-adaptin) , Proteintech , Cat#15690-1-AP.

Techniques: Marker, Recombinant, Lysis, Blocking Assay, Protease Inhibitor, Transfection, Bicinchoninic Acid Protein Assay, Derivative Assay, Plasmid Preparation, Software, Imaging

Journal: bioRxiv

Article Title: diaPASEF-Powered Chemoproteomics Enables Deep Kinome Interaction Profiling

doi: 10.1101/2024.11.22.624841

Figure Lengend Snippet: Re-defining the selection criteria for the CCA. (A) Parameter scanning experiment determining the CCA score (SCCA) for kinase PPIs predicted by our CCA in the SK-N-SH cell line. Increasingly stringent Student’s t-test log2 FC and p-value cut-off criteria were applied and only kinases or non-kinases fulfilling these criteria were used for CCA. Statistics: two sample Student’s t-test, range of log2 FC ≥ 0 to ≤ 5, and p ≥ 0 to ≤ 0.001. Refers to Fig. S2A . (B) Plotting the SCCA for varying log2 FC cut-offs and p-Value cut-offs against one another identified optimal mixed cut-off criteria for kinases input into our CCA. Refers to Fig. S2B . (C) Venn diagrams showing the overlap of reported kinase PPIs that were predicted by our 1 st gen CCA algorithm and PPIs predicted by our 2 nd gen CCA algorithm. Both 1 st and 2 nd gen CCA used the same diaPASEF kinobead AP-MS data from the two NB cell lines SK-N-SH and SH-SY5Y as the input. Refers to Table S1 . (D) Heatmap showing the %TDR for PPIs achieved by our 2 nd gen CCA separated into 0.05-unit intervals of the CCA Pearson’s r-value for the 1 st through 10 th predicted kinase interactor of a non-kinase protein. The %TDR reflects the percentage of BioGRID-reported divided by BioGRID-unreported PPIs. Refers to Fig. S2C . (E) STRING network for interaction partners of the kinases AAK1, BMP2K, and BCR, as predicted by 2 nd gen CCA. (F) Volcano plot showing the results of a co-IP/MS experiment using a specific antibody that binds AP2B1. Only proteins that are also members of the STRING interaction network in (E) are shown. Statistics: two sample Student’s t-test p < 0.05. Refers to Fig. S2E . (G) Venn diagrams comparing the total number of predicted kinase PPIs of 1 st predicted kinase vs. 1 st – 10 th predicted kinase, as was determined by 2 nd gen CCA in the NB cell lines SK-N-SH and SH-SY5Y.

Article Snippet: Antibodies used were AAK1 (E8M3P) Rabbit mAb (Cell Signaling Technology, CST, #61527) and beta 2 Adaptin (AP2B1) pAb (Novus Biologicals, # NBP3- 29580).

Techniques: Selection, Co-Immunoprecipitation Assay