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. 2023 Oct 18;18(10):e0289339. doi: 10.1371/journal.pone.0289339

Fig 6. N- and C-terminal tagged forms of Ste5 from ssa1D ssa2D strains have enhanced antibody accessibility.

Fig 6

A. STE5-Myc9-2μ WCE ips from wild type (WT) and ssa1D ssa2D extracts. Lanes 1–4: WT WCE 100 mg, 50 mg, 25 mg, 12.5 mg; lanes 5–8: ssa1D ssa2D WCE 100 mg, 50 mg, 25 mg, 12.5 mg. Lane 9 empty. Lanes 10–14: WT WCE IPs 500 mg, 200 mg and 100 mg, 50 mg. Lane 15 empty. Lanes 16–19: ssa1D ssa2D IP WCE 500 mg, 200 mg, 100 mg, 50 mg. Blot probed with 9E10 antibody. B. STE5-MYC9-CEN WCE ips from WT and ssa1D ssa2D extracts. 0.2 mg WCE was IP’d with 9E10 and compared to 50 μg WCE. Blot was probed with 9E10. Lane 1: WT STE5-MYC9 WCE. Lane 2: ssa1D ssa2D STE5-MYC9 WCE. Lane 3: WT Ste5-Myc9 IP. Lane 5-Myc9 IP. Lane 4: ssa1D ssa2D Ste5-Myc9 IP. C. HA3-STE5-2μ WCE ips from WT and ssa1D ssa2D extracts. 12CA5 IPs were done with 0.5 mg of WCE and run next to 0.1 mg WCE. Blot was probed with 12CA5. Lane 1: WT Ste5-Myc9 WCE IP with 12CA5. Lane 2: WT HA3-Ste5 WCE IP with 12CA5. Lane 3: ssa1D ssa2D HA3-Ste5 WCE IP with 12CA5. Lane 4 empty. Lane 5: WT HA3-Ste5 WCE. Lane 6: ssa1D ssa2D HA3-Ste5 WCE. For A-C 0.5mg whole cell extract was IP’d with either 12CA5 or 9E10 and then probed with 9E10 in the immunoblot. D. Oligomerization between Ste5-Myc9 (pRM01) and HA3-Ste5-Myc9 (pSKM26) in WT and ssa1D ssa2D whole cell extracts. Lane 1: WCE WT Ste5-Myc9. Lane 2: WCE WT HA3-Ste5. Lane 3: Empty. Lane 4: IP 9E10, WT Ste5-Myc9 + HA3-Ste5. Lane 5: IP 9E10, ssa1D ssa2D Ste5-Myc9 + HA3-Ste5. Lane 6: Empty. Lane 7: IP 12CA5 WT Ste5-Myc9 + HA3-Ste5. Lane 8: IP 12CA5 ssa1D ssa2D Ste5-Myc9 + HA3-Ste5. Lane 9: Empty. Lane 10: WCE WT Ste5-Myc9. Lane 11: WCE ssa1D ssa2D Ste5-Myc9. For A-D, extracts were prepared from WT and ssa1D ssa2D strains (EY3136, EY3141) expressing STE5-MYC9 2μ (pSKM19), HA3-STE5 (pSKM87) or STE5-MYC9 CEN (pSKM12) grown at 30°C in SC-uracil medium containing 2% dextrose.