Anti mcherry ab125096

For Phos-tag assay, the FgHapX-mCherry fusion construct was transferred into the ΔFgHapX and ΔFgYak1 mutants and the FgHapX^S245A/S338A-mCherry fusion construct was transferred into the ΔFgHapX mutant. Protein samples were prepared and extracted as described above. Each resulting protein sample was loaded on 8% SDS-polyacrylamide gels prepared with 25 μM Phos binding reagent acrylamide (APE×BIO, F4002) and 100 μM ZnCl₂. Gels were electrophoresed at 20 mA/gel for 3–5 h. Prior to protein transfer, gels were first equilibrated three times in transfer buffer containing 5 mM EDTA for 5 min and further equilibrated in transfer buffer without EDTA for 5 min for two times. Protein transfer from the Zn²⁺-phos-tag acrylamide gel to the PVDF membrane was performed for 4–5 h at 100 V on ice, and finally the membrane was analyzed by western blotting with the monoclonal anti-mCherry ab125096 (Abcam, Cambridge, UK) antibody.

Lysates used for immunoblotting were prepared in ice-cold RIPA Buffer containing HALT protease inhibitors (ThermoFisher). Clarified cell lysates were resolved on 4–15% mini-PROTEAN TGX pre-cast gels (Bio-Rad), and transferred to nitrocellulose membranes using a TransBlot Turbo (Bio-Rad, Hercules, CA, USA). Membranes were blocked with Odyssey TBS Blocking Buffer (Li-Cor, Licoln, NE, USA) and then probed with the appropriate antibodies. Primary antibodies were diluted in TBS + 1% PVP as follows: anti-PDX1, ab47267 1:5000 (Abcam, Cambridge, MA, USA); anti-PDX1, sc-14664, 1:5000 (Santa Cruz, Dallas, TX, USA) anti-Tubulin, T5326, 1:20000 (Sigma); anti-βactin, A5316, 1:5000 (Sigma); anti-eGFP, ab290, 1:5000 (Abcam); anti-HA, ab9110, 1:5000 (Abcam); anti-mCherry, ab125096, 1:2000 (Abcam) anti-OLLAS; NBP1-06713, 1:1000 (Novus, New York, NY); anti-FLAG, F3165-.2MG, 1:400 (Sigma); anti-His, 12698, 1:1000 (CST, Danvers, MA); anti-myc, 2278, 1:1000 (CST), anti-V5, MA5-15253, 1:1000 (ThermoFisher). Membranes were incubated with appropriate antibodies overnight at 4°C. Secondary antibodies (Li-Cor) were diluted 1:10 000 in Odyssey TBS Blocking Buffer (Li-Cor) with 0.2% Tween 20 added and incubated with membranes for 1 h at room temperature. Immunoblots were developed using a Li-Cor Odyssey CLx.

FgYak1-Flag and FgHapX-mCherry were co-transformed into the wild type. The nuclei of corresponding strains were extracted as described previously [100 (link)] and then fixed on the polylysine slides. Slides were incubated with the polyclonal anti-Flag antibody A9044 (Sigma, St. Louis, MO) and monoclonal anti-mCherry ab125096 (Abcam, Cambridge, UK) antibody at 1:500 dilution, respectively, followed by the secondary antibodies Andy Fluor 594 goat anti-mouse L119A (red fluorescence) (GeneCopoeia, Maryland, US) and Andy Fluor 488 goat anti-rabbit L110A (green fluorescence) (GeneCopoeia, Maryland, US) at 1:300 dilution. Nuclei were stained with DAPI for 15 minutes before fluorescence observation.