Anti pcofilin ser3

A375, A375-GFP-PMCA4b, A375-GFP-PMCA4b-LA, MCF-7, MCF-7-GFP-4b, and MCF-7-Sh-PMCA4b cells were cultured in a 6-well plate for 48 h. The total protein content of the cells was precipitated with 6% TCA. Samples were separated by using 10% or 15% acrylamide gels, as appropriate, and electroblotted onto PVDF membranes (Biorad, Hercules, CA, USA), as described previously [28 (link)].
Blots were immunostained with the following rabbit monoclonal primary antibodies: antivinculin (1:100, ThermoFisher scientific, cat. # 700062), anti-P-cofilin (Ser3) (1:1000, Cell Signaling Technology, Danvers, MA, USA, cat. # 77G2), rabbit polyclonal antibody: anti-β-tubulin (1:1000, Abcam, cat. # ab6046), anti-PMCA1 (1:1000, Affinity BioReagents, cat. # PA1-914), mouse monoclonal antibodies: anti-PMCA4 (JA9, 1:1000, Sigma-Aldrich, cat. # P1494), anti-NA⁺/K⁺ ATPase (1:2000, Enzo Life Sciences, cat. # BML-SA247), and chicken polyclonal antibody: anti-GFP (1:5000, Aves, GFP-1020). Horseradish peroxidase-conjugated anti-rabbit, anti-mouse, or anti-chicken secondary antibodies were used for detection (Jackson ImmunoResearch, dilution 1: 10,000) and were visualized with Pierce ECL Western Blotting Substrate (Thermo Scientific). The ImageJ software was used for densitometry analysis.

anti-GFP (chicken polyclonal IgY, GFP-1010, Aves Labs, Tigard, OR, USA; 1:2000 for ICC), anti-GFP (rabbit polyclonal, GTX113617, GeneTex, Irvine, CA, USA; 1:2000 for WB), anti-GFP (rabbit polyclonal, NB600-308, Novus Biologicals, Littleton, CO, USA; 1:500 for IP), anti-DDDDK (Flag) (mouse monoclonal, FLA-1, MBL Life Sciences, Tokyo, Japan; 1:10000 for ICC and WB), anti-β-actin (mouse monoclonal, 2D4H5, Proteintech, Rosemont, IL, USA; 1:10000 for WB), anti-α-tubulin (rabbit polyclonal, #11224-1-AP, Proteintech; 1:5000 for WB), anti-TDP-43 (rabbit polyclonal, RN107PW, MBL; 1:1000 for WB), anti-FMP1 (rabbit polyclonal, RN016P, MBL; 1:1000 for WB), anti-PSD95 (mouse monoclonal, 6G6-1C9, Abcam, Cambridge, UK; 1:500 for ICC, 1:3000 for WB), anti-pCofilin (Ser3) (rabbit monoclonal, 77G2, Cell Signaling Technology, Danvers, MA, USA; 1:1000 for WB), anti-pLIMK1 (Thr508)/LIMK2 (Thr505) (rabbit polyclonal, #3841, Cell Signaling Technology; 1:1000 for WB), anti-MAP2(chicken polyclonal IgY, Poly28225, Biolegend, San Diego, CA, USA; 1:10000 for ICC), anti-SMI312 (mouse monoclonal, 837904, Biolegend; 1:1000 for ICC), and anti-Homer1 (rabbit polyclonal, GTX103278, GeneTex; 1:500 for ICC).

Free-floating mouse brain tissue sections were carried through antigen retrieval in citric acid buffer (0.1 M citric acid, 0.2 M Na₂HPO₄) heated to 80°C for 30 min, and incubated in the rabbit primary antibody anti-pCofilin (Ser3) (cat#3311, Cell Signaling) (1:1000 μl) or rabbit anti-GH1 (Protein Tech Lab, cat# 55243–1-AP) (1:500 μl) for 48 h at 4°C, and subsequently in biotinylated secondary antibody (goat anti-rabbit IgG; 1:500; Vector Labs, Inc. Burlingame, CA), followed by streptavidin conjugated with horse-radish peroxidase for 2 h (1:5000 μl, Zymed, San Francisco, CA), and, finally, in nickel-enhanced diaminobenzidine/ peroxidase reaction (0.02% diaminobenzidine, Sigma-Aldrich, 0.08% nickel-sulfate, 0.006% hydrogen peroxide in PB). All solutions were made in PBS with 0.2% Triton X (PBS-Tx) unless otherwise specified. Immunostained sections were mounted on gelatin-coated glass slides, dehydrated in a gradient ethanol series, coverslipped, and coded for blinded quantitative analysis. All sections included in the study were processed simultaneously within the same session to avoid procedural differences. Omission of the primary or secondary antibodies did not result in detectable signal.