anti hook3 antibody  (Proteintech)

Journal: The Journal of Cell Biology

Article Title: Single-molecule imaging of stochastic interactions that drive dynein activation and cargo movement in cells

doi: 10.1083/jcb.202210026

Figure Lengend Snippet: Dextran as endosomal cargo marker and EGFR’s interaction with Hook proteins. (a) Spinning disk microscopy images of mCherry-Rab5 (left) and dextran-A647 (right). The signal-to-noise ratio was higher in cells with endocytosed dextran, allowing us to track dextran vesicles with high spatio-temporal resolution and perform dual channel imaging along with single molecules of dynein. (b) Spinning disk microscopy image from a 60-s-long time-lapse video of mCherry-Rab5 (green) and dextran-A647 (magenta) in cells (left) and the corresponding kymograph (right). Yellow arrowheads point to colocalized Rab5 and dextran, indicating dextran vesicles were a proxy for early endosomal compartments. In images acquired within 60 min after a 10-min pulse of dextran, 63 ± 14% of the dextran vesicles were associated with a Rab5 punctae ( n = 1 25 dextran vesicles from n = 1 independent experiment with 17 cells). (c) Immunofluorescence images of EGFR (left, green), Hook1 (middle, magenta), and their merge (right) obtained using Airyscan confocal microscopy. The inset (marked with a white box) is depicted at the bottom of the images. EGFR and Hook3 channel insets are depicted as intensity maps and the white arrowheads point to EGFR punctae, some of which colocalize with Hook1. (d) Immunofluorescence images of EGFR (left, green), Hook3 (middle, magenta), and their merge (right) obtained using Airyscan confocal microscopy. The inset (marked with a white box) is depicted at the bottom of the images. EGFR and Hook3 channel insets are depicted as intensity maps and the white arrowheads point to EGFR punctae, some of which colocalize with Hook3. (e) Plot of the probability of co-occurrence of EGFR with Hook1 and Hook3, showing a slightly higher probability of Hook1 being found on EGFR vesicles compared with Hook3. We confirmed that the colocalization of EGFR with Hook1 and Hook3 was not coincidental by calculating the probability of co-occurrence after flipping the EGFR channel horizontally and proceeding with our analysis. For both Hook1 and Hook3, the colocalization probability with EGFR reduced significantly with the flipped image (flipped EGFR with Hook1: 0.3 ± 0.2 [mean ± SD]; with Hook3: 0.2 ± 0.1 [mean ± SD]; both P < 10 −4 two-sample Kolmogorov–Smirnov test), indicating that the colocalization probability calculated from the original image is a true representation. n = ∼25 cells across three independent experiments. (f) Spinning disk microscopy image from a 10-s-long time-lapse video of dextran-A647 in cells treated with 10 µM nocodazole (left) and the corresponding kymograph (right). The kymograph shows abrogation of directed transport, as expected, upon MT depolymerization. (g) Mean squared displacement (MSD) analysis of dextran vesicles tracked in cells treated with 10 µM nocodazole for >30 min. The MSD data of dextran vesicles was fit to = 4Dt + c , and the intercept c was estimated to 0.0008. The diffusion coefficient D was 0.003 µm 2 /s, indicating that even in the absence of MTs, intracellular crowding likely prevented the dextran vesicles from diffusing away. ( n = 804 dextran vesicles from n = 1 independent experiment with 24 cells.) Error bars represent SEM. (h) Histogram of net movement of dextran (gray) and EGF (brown) endosomes, indicating that EGF-containing endosomes undertook more net minus end–directed movements in these 3-min time-lapse videos. (i) Probability distribution ( Ρ + (τ) ) of the plus end–directed runs for dextran (gray) and EGF (brown) vesicles. The plus end run time for both dextran and EGF were calculated to be 0.6 ± 0.2 s. (j) Quantification of the mean pixel intensity of Hook1 (light gray) and Hook3 (dark gray) in NC and Hook1/Hook3 siRNA cells. siRNA of Hook1 resulted in a reduction of Hook1 by 33.9% and reduction of Hook3 by 37.9% ( n > 100 cells across n = 2 independent experiments). (k) Representative images of EGF in control cells (left, “NC”), and cells with Hook1 siRNA (middle) and Hook3 siRNA (right), fixed 20 min after the addition of fluorescent EGF. Dashed lines indicate cell boundaries. (l) Plots of mean dispersion of EGF vesicles (left) and displacement between the center of mass of EGF vesicles and the cell centroid (right) in NC, Hook1 siRNA, and Hook3 siRNA cells. “n.s.” represents no significant difference and ** represents P < 0.01 ( n > 45 cells across n = 3 independent experiments, Kruskal–Wallis test). In a, b, f, and k, “N” marks the location/direction of the nucleus. Error bars in e, j, and l represent SD.

Article Snippet: The following primary antibodies were used: Rabbit DYNC1H1 Polyclonal Antibody (Cat# PA5-68173, RRID:AB_2691896, 1 µg/ml; Thermo Fisher Scientific), Rabbit Dynactin 1 Polyclonal Antibody (Cat# PA5-21289, RRID:AB_11155448, 2 µg/ml; Thermo Fisher Scientific), Mouse α Tubulin Monoclonal Antibody (Cat# 32-2500, RRID:AB_2533071, 2 µg/ml; Thermo Fisher Scientific), Rabbit anti-beta Tubulin antibody directly conjugated to AlexaFluor405 (Cat# ab179513, 250 µg/ml, RRID:AB_3073861; Abcam), Rabbit anti-Hook1 (Cat# ab151756, RRID:AB_3076228; Abcam), Rabbit anti-Hook3 (Cat# HPA024756, RRID:AB_1850913; Sigma-Aldrich), Mouse anti-EGFR (Cat# ab30, RRID:AB_303483; Abcam).

Techniques: Marker, Microscopy, Imaging, Immunofluorescence, Confocal Microscopy, Diffusion-based Assay, Dispersion

Journal: Molecular Biology of the Cell

Article Title: Rab11 regulates autophagy at dendritic spines in an mTOR- and NMDA-dependent manner

doi: 10.1091/mbc.e23-02-0060

Figure Lengend Snippet: FIGURE 2: mTOR inhibition targets Rab11 to Atg9A-positive reservoirs. (A) Representative Airyscan images of dendrites of neurons that were transfected with a plasmid encoding EGFP (blue) on DIV22 to allow accurate dendritic spine tracing and immunostained the next day for native Rab11 (magenta) and Atg9A, Hook3, or syntaxin 12 (stx12; green). The overlay channel shows Rab11 (magenta), Atg9A, Hook3, or stx12 (green), and the EGFP channel as an outline. The plasmid was expressed overnight. The neurons were incubated with INK128 (300 nM) for 20 min, fixed, and immunostained. Scale bar = 2.5 µm. The rightmost photomicrographs represent individual dendritic spines indicated in

Article Snippet: For Western blot (WB), immunofluorescence (IF), and immunoprecipitation (IP), the following primary antibodies were used: rabbit antiphospho-Akt (Ser473; Cell Signaling Technology, catalogue no. 4060, 1:1000 for WB), mouse anti-Akt (Cell Signaling, catalogue no. 2920, 1:1000 for WB), rabbit anti-Atg9A antibody (Thermo Fisher Scientific, catalogue no. PA5-21043, 1:200 for IF, 1:100 for WB), rabbit anti-caspase 3 antibody (Cell Signaling, catalogue no. 9662; 1:1000 for WB), mouse monoclonal anti-FLAG(R) M2 antibody (Sigma, F1804, 1:200 for IF) rabbit anti-phospho-GluA1 (Ser845; Millipore, catalogue no. 04-1073, 1:500 for WB), mouse anti-GluA1 (Santa Cruz Biotechnology, catalogue no. sc-55509, 1:100 for WB), rabbit anti-Hook3 antibody (Proteintech, catalogue no. 15457-1-AP, 1:100 for IF), rabbit anti-LC3B antibody (Thermo Fisher Scientific, catalogue no. PA1-46286, 1:200 for WB), rabbit anti-LC3B antibody (Sigma Aldrich, catalogue no. L7543, 1:2000 for WB), rabbit antiSQSTM1/p62 polyclonal antibody (Cell Signaling, catalogue no. 9662; 1:1000 for WB), rabbit anti-syntaxin 12 antibody (Proteintech, catalogue no. 14259-1-AP, 1:100 for IF), mouse anti-Rab11 antibody (BD Biosciences, catalogue no. 610656, Clone 47/Rab11, 1:50 for IF), and rabbit anti-Rab11a antibody (Thermo Fisher Scientific, catalogue no. 71-5300, 5 μg for IP), rabbit anti-phospho ribosomal protein S6 (Ser235/236; Cell Signaling Technology, catalogue no. 4858, 1:1000 for WB), mouse antiribosomal protein S6 (Cell Signaling Technology, catalogue no. 2317, 1:1000 for WB), mouse anti-αtubulin (Sigma-Aldrich, catalogue no. T5168, 1:5000 for WB).

Techniques: Inhibition, Transfection, Plasmid Preparation, Incubation

Journal: Cell Death Discovery

Article Title: HOOK3 suppresses proliferation and metastasis in gastric cancer via the SP1/VEGFA axis

doi: 10.1038/s41420-024-01808-8

Figure Lengend Snippet: A Total of 81 GC samples and 70 normal adjacent tissues were subjected to immunohistochemistry (IHC) analysis to assess the expression of HOOK3. An illustrative image representing the results is provided. B The overall survival of GC patients was stratified based on high and low HOOK3 expression groups, and Kaplan-Meier curves were generated. C Multivariate Cox regression analysis was conducted to evaluate the association between HOOK3 expression and overall survival, as depicted in the forest plots. D Western blotting was employed to measure HOOK3 protein expression in GES-1, AGS, HGC27, MKN28, and MKN45 cells, with GAPDH serving as a loading control. The Image J program was utilized for the quantification of Western blot band densities. The resulting values were presented as means with standard deviations (SD), and statistical significance was evaluated using Student’s t -test. ** P < 0.01, and *** P < 0.001.

Article Snippet: The Western blotting procedure employed a selection of primary antibodies, namely HOOK3 (Proteintech, #15457-1-AP), VEGFA (Proteintech, #19003-1-AP), SP1 (Proteintech, #21962-1-AP), and GAPDH (Proteintech, #60004-1-Ig).

Techniques: Immunohistochemistry, Expressing, Generated, Western Blot, Control

Journal: Cell Death Discovery

Article Title: HOOK3 suppresses proliferation and metastasis in gastric cancer via the SP1/VEGFA axis

doi: 10.1038/s41420-024-01808-8

Figure Lengend Snippet: HOOK3 expression and clinical features in 81 gastric cancer patient samples.

Article Snippet: The Western blotting procedure employed a selection of primary antibodies, namely HOOK3 (Proteintech, #15457-1-AP), VEGFA (Proteintech, #19003-1-AP), SP1 (Proteintech, #21962-1-AP), and GAPDH (Proteintech, #60004-1-Ig).

Techniques: Expressing

Journal: Cell Death Discovery

Article Title: HOOK3 suppresses proliferation and metastasis in gastric cancer via the SP1/VEGFA axis

doi: 10.1038/s41420-024-01808-8

Figure Lengend Snippet: A The protein expression of HOOK3 in MKN-28 and AGS cells was examined following transfection with si-HOOK3-1 or si-HOOK3-2. GAPDH was used as a loading control. The densities of Western blot bands were quantified using the ImageJ program. B The proliferation of AGS and MKN-28 cells was assessed after transfection with si-HOOK3-1 or si-HOOK3-2 using a CCK-8 assay. C Colony formation assays were performed on AGS and MKN-28 cells transfected with si-HOOK3-1 or si-HOOK3-2. D EdU analysis was conducted to evaluate the proliferative ability of AGS and MKN-28 cells treated with si-HOOK3-1 or si-HOOK3-2. Representative images were provided, and a scale bar of 100 μm was included. The bar graph shows the statistical analysis of the percentage of EdU-positive cells in transfected GC cells. E Transwell migration and invasion assays were conducted on AGS and MKN-28 cells transfected with si-HOOK3-1 or si-HOOK3-2. F Apoptosis assays were conducted were performed on AGS and MKN-28 cells transfected with si-HOOK3-1 or si-HOOK3-2. The experiments were performed in triplicate. The data are presented as the mean with standard deviation (SD), and statistical significance was determined using Student’s t -test. * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: The Western blotting procedure employed a selection of primary antibodies, namely HOOK3 (Proteintech, #15457-1-AP), VEGFA (Proteintech, #19003-1-AP), SP1 (Proteintech, #21962-1-AP), and GAPDH (Proteintech, #60004-1-Ig).

Techniques: Expressing, Transfection, Control, Western Blot, CCK-8 Assay, Migration, Standard Deviation

Journal: Cell Death Discovery

Article Title: HOOK3 suppresses proliferation and metastasis in gastric cancer via the SP1/VEGFA axis

doi: 10.1038/s41420-024-01808-8

Figure Lengend Snippet: A The protein expression of HOOK3 was examined in MKN-28 and HGC-27 cells with stable overexpression of HOOK3. GAPDH was used as a loading control. The quantification of band densities in Western blot analysis was performed using the ImageJ program. B The proliferation of MKN-28 and HGC-27 cells with stable overexpression of HOOK3 was assessed using a CCK-8 assay. C The colony formation assay was conducted to evaluate the ability of HOOK3 overexpressing MKN-28 and HGC-27 cells to form colonies. D EdU analysis was carried out to measure the proliferative ability of MKN-28 and HGC-27 cells with HOOK3 overexpression. Representative images are presented, with a scale bar of 100 μm. The percentage of EdU-positive cells in transfected GC cells was statistically analyzed and shown in the bar graph. E Transwell migration and invasion assays were performed to examine the migratory and invasive capacities of MKN-28 and HGC-27 cells with HOOK3 overexpression. F Apoptosis assays were performed on MKN-28 and HGC-27 cells with HOOK3 overexpression. The experiments were conducted in triplicate. The data were represented as means with standard deviation (SD), and statistical significance was assessed using Student’s t -test. ** P < 0.01, and *** P < 0.001.

Article Snippet: The Western blotting procedure employed a selection of primary antibodies, namely HOOK3 (Proteintech, #15457-1-AP), VEGFA (Proteintech, #19003-1-AP), SP1 (Proteintech, #21962-1-AP), and GAPDH (Proteintech, #60004-1-Ig).

Techniques: Expressing, Over Expression, Control, Western Blot, CCK-8 Assay, Colony Assay, Transfection, Migration, Standard Deviation

Journal: Cell Death Discovery

Article Title: HOOK3 suppresses proliferation and metastasis in gastric cancer via the SP1/VEGFA axis

doi: 10.1038/s41420-024-01808-8

Figure Lengend Snippet: A Volcano plot of differentially expressed genes (DEGs) in MKN-28 cells and HOOK3-knockdown MKN-28 cells. B Gene Ontology (GO) enrichment analysis of the top 0.5% negatively correlated genes among the DEGs. C The frequency of genes related to migration and invasion observed in the GO enrichment analysis. D The protein levels of VEGFA were measured in HOOK3-overexpressing MKN-28 and HGC-27 cells, as well as HOOK3-knockdown MKN-28 and HGC-27 cells, with GAPDH serving as a loading control. E The protein levels of VEGFA were assessed in HOOK3-overexpressing MKN-28 and HGC-27 cells treated with a plasmid that overexpresses VEGFA, with GAPDH used as a loading control. F The proliferation of MKN-28 and HGC-27 cells overexpressing HOOK3 and treated with a plasmid overexpressing VEGFA was evaluated using a CCK-8 assay. G Colony formation assays were performed on MKN-28 and HGC-27 cells with HOOK3 overexpression and treated with a VEGFA-overexpressing plasmid. H EdU analysis was conducted on MKN-28 and HGC-27 cells overexpressing HOOK3 after treatment with a plasmid that overexpresses VEGFA. I Transwell migration and invasion assays were carried out on HOOK3-overexpressing MKN-28 and HGC-27 cells treated with a VEGFA-overexpressing plasmid. The experiments were conducted in triplicate. The values were represented as means with standard deviation (SD), and statistical significance was determined using Student’s t -test. * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: The Western blotting procedure employed a selection of primary antibodies, namely HOOK3 (Proteintech, #15457-1-AP), VEGFA (Proteintech, #19003-1-AP), SP1 (Proteintech, #21962-1-AP), and GAPDH (Proteintech, #60004-1-Ig).

Techniques: Knockdown, Migration, Control, Plasmid Preparation, CCK-8 Assay, Over Expression, Standard Deviation

Journal: Cell Death Discovery

Article Title: HOOK3 suppresses proliferation and metastasis in gastric cancer via the SP1/VEGFA axis

doi: 10.1038/s41420-024-01808-8

Figure Lengend Snippet: A The intersection of transcription factors predicted by three databases, GTRD (red circle), JASPAR (green circle), and TFDB (blue circle), was analyzed. B The expression of YY1, SP1, and ZEB1 was analyzed by RT-qPCR in HOOK3 knockdown MKN-28 and HGC-27 cells. C The expression of YY1, SP1, and ZEB1 was analyzed by RT-qPCR in HOOK3-overexpressing MKN-28 and HGC-27 cells. D Western blot analysis was performed to evaluate the expression of SP1 in either HOOK3 knockdown or HOOK3-overexpressing MKN-28 cells. GAPDH was used as the control. The band densities from Western blot were quantified using the ImageJ program. E Western blot analysis was performed to evaluate the expression of SP1 in either HOOK3-knockdown or HOOK3-overexpressing HGC-27 cells. GAPDH was used as the control. The band densities from Western blot were quantified using the ImageJ program. F A schematic diagram illustrating the presence of three SP1 binding sites (P1: −367nt to −358nt, P2: −642nt to −632nt, and P3: −76nt to −68nt) in the 5′ region of the VEGFA promoter. G ChIP analysis was conducted to assess the binding of SP1 to the VEGFA promoter in MKN-28 cells. Normal rabbit IgG was used as the control. H The luciferase activity, responsive to SP1, was measured in MKN-28 and HGC-27 cells transfected with HOOK3 overexpressing plasmid and SP1 overexpressing plasmid. I Western blot analysis was performed to evaluate the expression of VEGFA and SP1 in HOOK3-overexpressing MKN-28 cells transfected with SP1 overexpression plasmid. GAPDH was used as the control. The band densities from Western blot were quantified using the ImageJ program. J Western blot analysis was performed to evaluate the expression of VEGFA and SP1 in HOOK3-overexpressing HGC-27 cells transfected with SP1 overexpression plasmid. GAPDH was used as the control. The band densities from Western blot were quantified using the ImageJ program. The experiments were conducted in triplicate. The mean values with standard deviation (SD) were presented, and the statistical significance was determined using Student’s t -test. Nonsignificant results were denoted as “ns”, while significance levels were shown as * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: The Western blotting procedure employed a selection of primary antibodies, namely HOOK3 (Proteintech, #15457-1-AP), VEGFA (Proteintech, #19003-1-AP), SP1 (Proteintech, #21962-1-AP), and GAPDH (Proteintech, #60004-1-Ig).

Techniques: Expressing, Quantitative RT-PCR, Knockdown, Western Blot, Control, Binding Assay, Luciferase, Activity Assay, Transfection, Plasmid Preparation, Over Expression, Standard Deviation

Journal: Cell Death Discovery

Article Title: HOOK3 suppresses proliferation and metastasis in gastric cancer via the SP1/VEGFA axis

doi: 10.1038/s41420-024-01808-8

Figure Lengend Snippet: A The image displayed is representative of HOOK3-overexpressing MKN28 tumors grown in nude mice. The volumes of these tumors were evaluated in groups containing four mice each. B Representative images of immunohistochemistry (IHC) staining for Ki-67 and VEGFA and TUNEL staining were captured in tumor tissues from the OE-HOOK3 and OE-NC groups. The scale bar represents 50 μm. C A murine lung metastasis model was established using HOOK3-overexpressing GC cells and control GC cells, with three mice in each group. D Representative images of hematoxylin and eosin (HE) stains were obtained from lung metastasis in the OE-HOOK3 and OE-NC groups. The scale bar represents 50 μm. The mean values along with standard deviations (SD) were used to represent the data, and statistical significance was determined using Student’s t -test. ** P < 0.01.

Article Snippet: The Western blotting procedure employed a selection of primary antibodies, namely HOOK3 (Proteintech, #15457-1-AP), VEGFA (Proteintech, #19003-1-AP), SP1 (Proteintech, #21962-1-AP), and GAPDH (Proteintech, #60004-1-Ig).

Techniques: Immunohistochemistry, TUNEL Assay, Staining, Control

Journal: Cell Death Discovery

Article Title: HOOK3 suppresses proliferation and metastasis in gastric cancer via the SP1/VEGFA axis

doi: 10.1038/s41420-024-01808-8

Figure Lengend Snippet: Schematic diagram of regulation mechanism of HOOK3/VEGFA/SP1 axis in GC.

Article Snippet: The Western blotting procedure employed a selection of primary antibodies, namely HOOK3 (Proteintech, #15457-1-AP), VEGFA (Proteintech, #19003-1-AP), SP1 (Proteintech, #21962-1-AP), and GAPDH (Proteintech, #60004-1-Ig).

Techniques: