Alexa flour 488 conjugated anti rabbit igg

ZR75–1 cells were seeded onto glass coverslips in 6-well plates overnight and transfected with TAp73α for 48 hrs. Detached cells were harvested and fixed onto slides by using cytospin (Shandon, Inc). For mitochondria staining, 100nM mitotracker (M22425, Molecular Probes Inc) were added to the culture medium for 15 minutes before fixing with 4% paraformaldehyde in PBS for 30 minutes at room temperature (RT). Fixed cells were washed with PBS and permeabilized with 0.05% Tween-20 in PBS for 10 minutes. After washing with PBS, the coverslips were incubated with anti-p73 (Ab-4, Calbiochem), diluted in 5% BSA/PBS for 1 hr at RT. They were then washed twice with PBS, followed by incubation with Alexa Flour 488 conjugated anti-rabbit IgG (Molecular Probes Inc) for 30 minutes at RT. After washing 3X with PBS, the coverslips were mounted with Vectashield^® containing DAPI (Vector Labs, CA). Cells were examined under a Leica SpE confocal microscope.

Puromycin and G-418 were purchased from Cayman Chemical (Ann Arbor, Michigan, USA). AG1478, and U0126 were purchased from Selleck. The protein A/G agarose and EGF were purchased from Sigma (St louis, MO, USA). The following antibodies were used: anti-β-actin (#8457), anti-cleaved-caspase-3 (#9664), ani-caspase-3 (#9662), anti-cleaved PARP (#5625), anti-EGFR (#4267), anti-Erk1/2 (#4695), anti-phospho-Erk1/2 (#4370), anti-p38 (#8690), anti-phospho-p38 (#9211), anti-AKT(#9272), anti-phospho-AKT (Ser473) (#4060), anti-phospho-JNK (Thr183/Tyr185) (#9251), anti-JNK (#9252), anti-HA-Tag (#3724), anti-c-Cbl (#2747), anti-ubiquitin (P4D1) (#3936) from Cell Signaling Technology (Beverly, MA, USA); anti-Flag-Tag from Sigma-Aldrich (St Louis, MO, USA), anti-GAPDH (AC002), anti-phospho-EGFR (Y1068) (AP0820) from Abclonal Technology(Shanghai, China); anti-14-3-3σ (ab14123), anti-Bim (ab7888) from Abcam (Cambridge, MA, USA); anti-Bcl-2 (12789-1-AP) from proteintech (Chicago, IL, USA); Alexa Flour 488-conjugated anti- rabbit IgG, Alexa Flour 555-conjugated anti-rabbit IgG and Alexa Flour 555-conjugated anti-mouse IgG from Thermo Scientific (Rockford, IL, USA). HRP conjugated anti-rabbit IgG, HRP conjugated anti-mouse IgG from Beyotime Institute of Biotechnology; APC anti-human EGFR antibody (#352905) used for cell surface immunofluorescence staining from BioLegend (San Diego, CA).

Immunofluorescence (IF) was performed as previously described^{43 (link)}. Cells grown on coverslip were fixed with 1% pFA in PBS containing 200 mM sucrose for 10 min. Cells were washed three times with 1 ml PBS and treated with 0.5% Triton-X100 in PBS at 4 °C for 5 min. Cells were again washed three times with 1 ml PBST (PBS containing 0.05% Tween 20). Cells were soaked in PBS containing 1% BSA at room temperature for 10 min and incubated for 30 min with 1:1000-diluted primary antibodies, such as mouse monoclonal anti-IL-1β (Santa Cruz Biotechnologies, E7-2-hIL1β), mouse monoclonal anti-p21 (Santa Cruz Biotechnologies, sc-817), mouse monoclonal anti-FLAG M2 (Sigma–Aldrich), rabbit polyclonal anti-Pol II (Bethyl Laboratories, A300-653A), rabbit polyclonal anti-Myc (Abcam, ab9106), and mouse monoclonal anti-Pol II (Covance, 8WG16). Cells were washed three times with 1 ml PBST and incubated with 1:1000-diluted secondary antibodies, Alexa Flour 488-conjugated anti-rabbit IgG (Thermo Fisher Scientific) and/or Cy3-conjugated anti-mouse IgG (Jackson ImmunoResearch) in PBS for 15 min. After washing cells twice with PBST, the coverslip was mounted onto slide glass using ProLong Gold antifade mountant (Thermo Fisher Scientific). Images were captured by a Zeiss Axioimager Z1 fluorescence microscope with an oil immersion objective lens (Plan Apochromat, 63 × , NA 1.4, Zeiss).

The cells were fixed using 4% paraformaldehyde (Wako) in PBS for 10 min, permeabilized with 0.5% Triton X-100 (Wako) for 30 min, and treated with 3% bovine serum albumin to block non-specific binding. Cells were further incubated with the primary antibodies for 16 h at 4°C followed by visualization using the appropriate secondary antibodies labeled with Alexa-488 and -594 with 4’, 6-diamidino-2-phenylindole (DAPI). The primary antibodies used include: mouse anti-myelin basic protein (MBP) (SMI-99, COVANCE); anti-βⅢ Tubulin (COVANCE); mouse anti-myelin associated glycoprotein (MAG), clone513 (Millipore); rabbit anti-neurofilament M (Merk). Alexa Flour 488-conjugated anti-rabbit IgG (Thermo Fisher Scientific) and Alexa Flour 594-conjugated anti-mouse IgG (Thermo Fisher Scientific) were used as secondary antibodies for immunocytochemistry. For quantification, myelination profiles visualized with MBP staining was quantified in 5 randomly selected fields using a 20x objective lens and the number of myelinated nerve fibers per arbitrary unit area was calculated.