Cells were fixed in 100% methanol on ice for 10 min, washed four times with PBS, and blocked in PBS containing 3% BSA, 0.1% Triton X‐100 (PBS‐TB) for 1 h at room temperature. Cells were incubated for 2 h with the indicated antibodies as the first antibodies, and for 1 h with Alexa Fluor 488‐conjugated anti‐rabbit or mouse IgG or Alexa Fluor 568‐conjugated anti‐rabbit or mouse IgG (Life Technologies) as the second antibodies with Hoechst 33342 (Life Technologies). Fluorescent images were obtained using a BZ‐9000 (Keyence, Osaka, Japan). The antibodies used were: multi‐ubiquitin (MBL, D058‐3), ubiquitin, Lys48‐Specific (Millipore; 05‐1307), TACC3 (Cell Signaling, 8069), LAMP2 (BD Biosciences, 555803), PMP70 (Abcam, Cambridge, UK; ab85550), GM130 (Abcam, ab52649), COX IV (Cell Signaling, 4850).
Tacc3
Manufactured by Cell Signaling Technology
Sourced in United Kingdom
TACC3 is a laboratory protein that is involved in the regulation of microtubule dynamics. It is a member of the transforming acidic coiled-coil (TACC) protein family and plays a role in the organization and stability of the mitotic spindle during cell division.
Automatically generated - may contain errors
Lab products found in correlation
Cox 4, by Cell Signaling Technology (1 mentions)
Lamp2, by BD (1 mentions)
Lys48 specific, by Merck Group (1 mentions)
Pmp70, by Abcam (1 mentions)
Gm130, by Abcam (1 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
Bz 9000, by Keyence (1 mentions)
Polyvinylidene difluoride membrane, by Thermo Fisher Scientific (1 mentions)
Enhanced chemi luminescence (ecl), by BD (1 mentions)
3 protocols using tacc3
1
Immunofluorescence Staining Protocol
2
Immunoblotting for p53 and TACC3
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Whole cell extracts were prepared by scraping cells in lysis buffer (150 mM NaCl, 5 mM EDTA, 0.5% NP40, 50 mM Tris, pH 7.5), resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride membranes (Thermo Fisher Scientific). Antibodies to p53 (DO-1, SC-126) and β-actin (C4, SC-47778) were from Santa Cruz. Antibodies phospho-TACC3 (S558, #8842) and TACC3 (#8069) were from Cell Signaling. Primary antibodies were detected with goat anti-mouse or goat anti-rabbit secondary antibodies conjugated to horseradish peroxidase (Life Technologies), using Clarity chemiluminescence (BIO-RAD).
3
Immunoblotting of GFP-PI3KC2α Variants
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HEK293T were transfected with plasmids encoding eGFP-PI3KC2α WT or mutants, mitotically arrested 24 h post-transfection and gathered as reported in ref. 26 (link). The following primary antibodies were used: TACC3 (Rabbit, no. 8069, Cell Signaling) and homemade GFP (Rabbit, polyclonal). Membranes probed with the indicated antibodies were then incubated with HRP-conjugated anti-Rabbit IgG light chain (1:5,000, 211-032-171, Jackson ImmunoResearch) and developed with enhanced chemiluminescence (ECL, BD).
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