β actin

The following primary antibodies were used: mouse monoclonal antibodies to α-tubulin (clone DM1A, IgG1, Invitrogen); β-tubulin (clone TBN06, Invitrogen); h-β-I-tubulin (IgG2b, R&D Systems, Minneapolis, MN, USA); h-β-III-tubulin (IgG2a, R&D Systems); pan-actin (clone C4, Cell Signaling Technology Inc., Danvers, MA, USA); β-actin (IgG1, MCA5775GA, AbD Serotec, Kidlington, UK); γ-actin (IgG2b, (MCA5776GA, AbD Serotec); β-catenin (IgG1, DAKO, Glostrup, Denmark); E-cadherin (IgG2a, BD Transduction); MDR-1 (Santa Cruz Biotechnology, Dallas, TX, USA); N-cadherin (Cell Signaling Technology Inc., Danvers, MA, USA); claudin-1 (Cell Signaling Technology Inc.); and vimentin (V9, DAKO).
The following secondary antibodies were used: AlexaFluor488-, Red-X, AlexaFluor594-conjugated goat anti-mouse IgG or specific to IgG1, IgG2b, IgG2a, absorbed to other IgG isotypes, and goat anti-rabbit IgG (Jackson ImmunoResearch Laboratories Inc., West Grove, PA, USA). DAPI (D9542, Sigma-Aldrich) was applied for DNA (nuclear) staining. HRP-conjugated secondary antibodies were used for Western blot analysis: anti-mouse IgG and anti-rabbit IgG (Santa Cruz Biotechnology).

β-Actin (MCA5775GA, AbD Serotec); γ-actin (MCA5776GA, AbD Serotec); p-p44/42 (T202/Y204) (4370S, Cell Signaling); ERK1/2 (4695S, Cell Signaling); p34-Arc (07-227, EMD Millipore); WAVE2 (3659P, Cell Signaling); Cofilin (D3F9) XP®, cofilin1 (5175P, Cell Signaling); p-Cofilin1 (Ser3) (3313, Cell Signaling); N-ras (sc-519, Santa Cruz); PP1α (S3010, Epitomics); Pan-actin (4968, Cell Signaling); α-tubulin (2144, Cell Signaling).
The following secondary Abs were used: FITC-, TRITC-, AlexaFluor488-, AlexaFluor594-, AlexaFluor647-, Cy5-conjugated goat anti-mouse IgG1, IgG2b, IgG and goat anti-rabbit IgG (Southern Biotechnology, Associates Inc., Birmingham, AL; Jackson; Life technologies).

Mouse monoclonal antibodies to: β-actin (MCA5775GA, AbD Serotec); γ-actin (MCA5776GA, AbD Serotec); pan actin (4968, Cell Signaling); α-tubulin (2144, Cell Signaling); rat monoclonal antibodies to α-tubulin (Serotec); rabbit polyclonal antibodies to EB1 (Gen Script) and tubulin (Sigma-Aldrich).
The following secondary antibodies were used: AlexaFluor488-, AlexaFluor594-, AlexaFluor647-, Cy5-conjugated goat anti-mouse IgG1, IgG2b, IgG, goat anti-rabbit IgG and goat anti-rat IgG (Southern Biotechnology, Associates Inc., Birmingham, AL; Jackson; Life technologies).

Mouse monoclonal antibodies to: β-actin (MCA5775GA, AbD Serotec); γ-actin (MCA5776GA, AbD Serotec, Raleigh, NC, USA); pan actin (4968, Cell Signaling) were used. Rabbit polyclonal antibodies to CENP-A (2186, Cell Signaling) were used. Rabbit monoclonal antibodies to: Phospho-Histone H3 (Ser10, 3377, Cell Signaling); Histone H3 (4499, Cell Signaling) were used.
The following secondary antibodies were used: AlexaFluor488-, AlexaFluor594- conjugated goat anti-mouse IgG, IgG1, IgG2b and goat anti-rabbit IgG (Jackson ImmunoResearch Laboratories Inc, West Grove, PA, USA).

Western blots were performed on whole brain and hippocampus as already described [50 (link),51 (link)]. Specific primary antibodies, i.e., anti-ikb-α (Santa Cruz Biotech, sc-1643), or anti-NFkB (Santa Cruz Biotechnology, sc-8008), or anti-Nrf2 (Santa Cruz Biotechnology, sc-36594), or anti-HO-1 (Santa Cruz Biotechnology, sc-136970), or anti-p-Tau (Santa Cruz Biotechnology, sc-32275). or anti-APP (Santa Cruz Biotechnology, sc-32277) were mixed in a 5% w/v nonfat dried milk solution and were incubated at 4 °C, overnight. Afterwards, blots were incubated with a peroxidase-conjugated bovine anti-mouse IgG secondary antibody or a peroxidase-conjugated goat antirabbit IgG (Jackson Immuno Research) for 1 h at room temperature [52 (link),53 (link)]. To verify the amounts of protein were equal, membranes were also incubated with an antibody against β-actin (Santa Cruz Biotechnology). Signals were detected with an enhanced chemiluminescence detection system reagent (Super-Signal West Pico Chemiluminescent Substrate, Pierce) [54 (link),55 (link)]. The relative expression of the protein bands was quantified by densitometry with Bio-Rad ChemiDoc XRS software and standardized to β-actin levels [56 (link)]. Images of blot signals were imported to an analysis software (Image Quant TL, v2003).

MDA-MB-231 and MCF-7 cells were lysed in RIPA lysis buffer on ice for 30 min, then centrifuged (12000 g/min; 30 min) at 4°C. A bicinchoninic acid (BCA) assay was used to detected protein concentrations. Equal amounts of total protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes (millipore, USA), and then incubated with primary antibodies overnight at 4°C after the membranes were blocked (5% skim milk in PBS with 0.1% Tween 20) for 4 h. The next day, the membranes were imaged with gel imaging equipment (Bio-Rad, USA) after the membranes were incubated with secondary antibodies for 2 h. β-actin was used as a loading control. The following antibodies were used: Bcl-2 and Bax (Cell Signaling technology, USA); anti-RIP1, anti-RIP3, and p-RIP3 (Santa Cruz Biotechnology, USA); TNF-α (Abcam, USA); Caspase 3 (Enzo, USA); Ppm1b (BETHYL, USA); anti-β-actin (Biosharp, China) All reagents were dissolved according to the manufacturer’s instructions.