Expression of dynamin-2, caveolin-1 and clathrin after their downregulation by specific siRNA or in A549-shCLTC and A549-shCAV-1 clones was assessed by Western blotting analysis. Cells (3 × 105 cells per sample) were lysed with Laemmli buffer heated to 95°C (150 μL/sample), scraped off the plate, sonicated and boiled (95°C, 3 min). Proteins were separated using 10% acrylamide gel by SDS-PAGE and transferred to nitrocellulose membrane (Amersham Protran, GE Healthcare, USA). The membrane was blocked with 5% (wt/vol) nonfat dry milk (Carl Roth, Germany) in Tris-Buffered Saline containing 1% Tween 20 and probed with the appropriate primary antibodies, followed by incubation appropriate horseradish peroxidase-conjugated IgG secondary antibody. Detection was performed with Pierce ECL Western Blotting Substrate (Thermo Fischer Scientific, USA), using ChemiDoc Imaging System (Bio-Rad, USA). Densitometry was performed with ImageJ software (NIH, USA). The following antibodies were used: anti CLTC (ab21679, Abcam, UK), anti CAV-1 (ab2910, Abcam, UK), anti DNM-2 (ab3457, Abcam, UK). Proteins were normalized to the total protein stained with amidoblack and the results were presented as relative expression of proteins compared to control cells.
Ab3457
Manufactured by Abcam
Sourced in United Kingdom, United States
Ab3457 is a laboratory equipment product offered by Abcam. It is designed to serve a core function without any interpretation or extrapolation on its intended use.
Automatically generated - may contain errors
Lab products found in correlation
Ab21679, by Abcam (2 mentions)
Ab2910, by Abcam (1 mentions)
Non fat dry milk, by Carl Roth (1 mentions)
Amersham protran, by GE Healthcare (1 mentions)
Pierce ecl western blotting substrate, by Thermo Fisher Scientific (1 mentions)
Chemidoc imaging system, by Bio-Rad (1 mentions)
Ab4069, by Abcam (1 mentions)
Ab150358, by Abcam (1 mentions)
Anti rabbit igg hrp, by Cell Signaling Technology (1 mentions)
Anti mouse igg hrp, by Cell Signaling Technology (1 mentions)
Alexafluor 488 goat anti rabbit igg a 11008, by Thermo Fisher Scientific (1 mentions)
Protein blocker, by Agilent Technologies (1 mentions)
Hematoxylin, by Agilent Technologies (1 mentions)
3 3 diaminobenzidine (dab), by Agilent Technologies (1 mentions)
Ab15200, by Abcam (1 mentions)
Ab11575, by Abcam (1 mentions)
Axiophot microscope, by Zeiss (1 mentions)
Alexa fluor 488 donkey anti rabbit secondary antibody, by Thermo Fisher Scientific (1 mentions)
Az100, by Nikon (1 mentions)
Vectashield mounting medium, by Vector Laboratories (1 mentions)
9 protocols using ab3457
1
Western Blot Analysis of Endocytic Proteins
2
Western Blot Analysis of Cell Signaling Proteins
3
Immunofluorescence Analysis of Caveolin-1
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Anti-clathrin heavy chain antibody (ab21679), anti-dynamin 2 antibody (ab3457) were obtained from Abcam. Caveolin-1 polyclonal antibody (PA5-17447), Alexa Fluor 488 goat anti-rabbit IgG (A11008) and horseradish peroxidase-conjugated donkey anti-rabbit IgG antibody (31458) were from Thermo Fisher Scientific.
4
Immunohistochemical Detection of Dynamin 2
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Briefly, TMA slides were deparaffinized, rehydrated, washed, and then endogenous peroxidase activity was blocked by 3 % H2O2 for 20 min at room temperature. Subsequently, the tissue slides were washed three times in Tris Buffered Saline (TBS). Also for antigen retrieval, the slides were autoclaved for 10 min in Tris-EDTA Buffer (pH = 9). After three times washing of the TMA slides in TBS, the slides were incubated for 20 min with 5 % sheep serum prepared in blocker protein (Dako, Denmark). Then, the slides were incubated overnight at 4 °C with the primary antibody (anti-dynamin 2 antibody, ab3457, Abcam, USA). Moreover, for isotype control, rabbit immunoglobulin (rabbit IgG) was used (both at a concentration of 100 ng/ml). After washing the slides, they were incubated for 1 h by TMMouse/Rabbit PolyVue HRP (DBS, USA) as the secondary antibody. Afterward, the slides were washed and then treated with 3,3’-diaminobenzidine (DAB) (Dako, Denmark) substrate as a chromogen for 20 min. After washing, the slides were counterstained with hematoxylin (Dako, Denmark). Finally, the slides were dehydrated, cleared in xylene, and then mounted.
5
Immunofluorescence Analysis of Muscle Tissue
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Cells and muscle tissue cryosections (8 μm thick) were fixed in paraformaldehyde 4% (15 min at room temperature). After washing in PBS, cells and cryosections were permeabilized in Triton X‐100 0.5% in PBS for 10 min at room temperature and blocked in PBS–Triton X‐100 0.1%, BSA 5% and donkey serum 5% for 30 min. Samples were incubated with rabbit anti‐Dynamin 2 (1:400 (Abcam ab3457), rabbit anti‐laminin (1:500, Abcam ab11575) or rabbit anti‐Desmin (1:200, ab15200) overnight at 4°C, in PBS with Triton X‐100 0.1% and BSA 1%. After PBS–Triton X‐100 0.1% washes, samples were incubated with donkey anti‐rabbit AlexaFluor488 secondary antibody (1:1,000, Life Technologies, France) for 60 min at room temperature. The slides were mounted with Vectashield mounting medium (Vector Laboratories, United Kingdom). Images were acquired using an axiophot microscope (Zeiss, France) or a macroscope (Nikon AZ100).
6
Cellular Localization of Immune Markers
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Antibodies to DC-STAMP1 (DCST1; Biorbyt, orb2242, rabbit polyclonal Ab), Dynamin (Abcam, ab3457, rabbit polyclonal Ab), CD9 (Abcam, ab223052, rabbit polyclonal Ab), Phalloidin-TRITC (Sigma-Aldrich, P1951), CD13 (SL13, Millipore, MABC950, rat monoclonal Ab).
7
Antibody Use in Protein Detection
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Antibodies were purchased from the indicated commercial sources: mouse anti-FLAG (M2, F3165, 1:1000-1:10000) and tubulin (Sigma-Aldrich, T6074, 1:1000-1:10000); mouse anti-Ku70 (sc-17789, 1:500), -Ku80 (sc-5280, 1:500), -DNA-PKcs (sc-1832, 1:1000), -Pol μ (sc-398666, 1:200), -PAXX (sc-514359, 1:20-1:100) and –XLF (sc-166488, 1:100-1:200) (all Santa Cruz Biotechnology); rabbit anti-PAXX (Atlas Antibodies, HPA045268, 1:1000), rabbit anti-PAXX (ab126353, 1:1000), Lig IV (ab80514, 1:1000) and dynamin-2 (all Abcam, ab3457, 1:100); rabbit anti-PAXX (D6X7 × , CST 92448 S), -XLF (CST 2854 S), -Artemis (CST 13381 S, 1:1000) and -γH2AX (all Cell Signalling Technology, CST 9718 S, 1:1000); mouse anti-XRCC4 (Thermofisher, MA5-24383), rabbit anti-Pol λ (Bethyl, A301-640S, 1:1000-1:10000; A301-641A, 1:1000-1:10000); rabbit anti-XRCC4 (1:500-1:1000) was a generous gift from Dr. Dik Van Gent (Erasmus University, Netherlands).
8
Antibody Validation and Ligand Stimulation
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Antibodies used were as follows: anti-Gulp1 (HPA020587; rabbit polyclonal; Sigma-Aldrich), anti-GFP (JL-8; mouse monoclonal; Clontech), anti-C-Myc (SC-40; mouse monoclonal; Santa Cruz), anti-β-tubulin (480011; mouse monoclonal; Thermo Fisher Scientific), anti-Dyn2 (ab3457; rabbit polyclonal; Abcam), M2 anti-FLAG (F3165; mouse monoclonal; Sigma-Aldrich), anti-FLAG (F7425; rabbit polyclonal; Sigma-Aldrich), and anti-HA (ab18181; mouse monoclonal; Abcam). Secondary antibodies purchased from Jackson ImmunoResearch Laboratories, including HRP-coupled secondary antibodies, were used for Western blots, and fluorescently labeled secondary antibodies were used for immunostaining. For STED imaging, Abberior Star 580 and Abberior Star Red secondary antibodies were purchased from Abberior Instruments.
For soluble ligand stimulation, Human IgG Fc fragment (Jackson ImmunoResearch Laboratories) or mouse ephrinB1-Fc fusion protein (R&D Systems) was incubated with goat anti-human Fc at a ratio of 5:1 for 30 min (Zimmer et al., 2003 (link)). Cells were incubated with the clustered proteins at a final concentration of 2 µg/ml for 30 min at 37°C.
Dynamin inhibitor Dyngo-4a (ab120689; Abcam) was diluted in DMSO.
For soluble ligand stimulation, Human IgG Fc fragment (Jackson ImmunoResearch Laboratories) or mouse ephrinB1-Fc fusion protein (R&D Systems) was incubated with goat anti-human Fc at a ratio of 5:1 for 30 min (Zimmer et al., 2003 (link)). Cells were incubated with the clustered proteins at a final concentration of 2 µg/ml for 30 min at 37°C.
Dynamin inhibitor Dyngo-4a (ab120689; Abcam) was diluted in DMSO.
9
Immunostaining and Immunoblotting Reagents
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Primary antibodies used in this study were rabbit polyclonal anti-dynamin 2 (ab3457, Abcam), rabbit polyclonal anti-PACSIN1 (MÀ46) (SC-30127, Santa Cruz), mouse monoclonal anti-PACSIN2 (SAB1402538, SIGMA), rabbit polyclonal anti-PACSIN2 [12] (link), rabbit polyclonal anti-PACSIN3 (AB37612, Abcam), GFP (D5.1) XP rabbit mAb (2956S, CST) and anti-DDDDK-tag pAb (PM020, MBL). Secondary antibodies and Alexa-conjugated phalloidin for immunofluorescence microscopy were purchased from Thermo Fisher Scientific: Alexa Fluor 350 goat anti-rabbit IgG (HþL) (A21068), Alexa Fluor 488 donkey anti-mouse IgG (HþL) (A21202), Alexa Fluor 488 donkey anti-rabbit IgG (HþL) (A21206), Alexa Fluor 555 donkey anti-mouse IgG (HþL) (A31570), Alexa Fluor 555 donkey anti-rabbit IgG (HþL) (A31572), Alexa Fluor 568 donkey anti-goat IgG (HþL) (A11057), Alexa Fluor 350 Phalloidin (A22281) and Alexa Fluor 555 Phalloidin (A34055). Secondary antibodies for immunoblot analyses were also purchased from Thermo Fisher Scientific: goat anti-rabbit IgG (HþL) secondary antibody, HRP (31460), rabbit anti-mouse IgG (HþL) secondary antibody, HRP (31450). Glutaraldehyde used for fixing gelatin coating coverslips was prepared from Glutaraldehyde 25% EM (G004, TAAB).
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