Skip to main content
. Author manuscript; available in PMC: 2020 Jan 17.
Published in final edited form as: Cell Chem Biol. 2018 Nov 15;26(1):85–97.e4. doi: 10.1016/j.chembiol.2018.10.009

Figure 4. Cys512 and Cys516 mutations restore IRP2 function under cisplatin treatment.

Figure 4

A) HA-tagged full length human IRP2 wild-type and Cys to Ala mutants (C512A, C516A, C512A/C516A, C578A/C581A) in pcDNA3.1/C-HA plasmids were stably transfected into SW480 cells. Expression of transfected HA-IRP2 was detected by Western blot with anti-HA antibody, and GAPDH as a loading control (WB, top). These cell lysates were also incubated with 20 µg/mL cisplatin and subjected to pull down assay using a biotin-FH IRE probe. IRP2 binding to IRE was detected by Western blot with anti-HA antibody (Pull down, bottom). B) SW480 cells stably transfected with IRP2 wt and mutants were treated with 10 µg/mL cisplatin for 36 h (top) or 100 µg/mL FAC for 16 h (bottom), and Western blots were performed with ferritin H (FH), TfR1 and GAPDH antibodies. C) SW48 cells stably transfected with empty vector, IRP2 (WT, C512A/C516A) and IRP1 (WT, C437S) were treated with 10 µg/ml cisplatin for 20 hr and Western blots were performed with ferritin H (FH), TfR1 and GAPDH antibodies. D) pcDNA3 (Empty), IRP2WT, or C512A/C516A expressing SW480 cell were treated with 25 µg/mL cisplatin for 22 h, subjected to RIP assay using anti-HA antibody, followed by qPCR using ferritin H (FH) or B2M primers. 10% of IP samples were subjected to Western blot with anti-HA antibody to check IP efficiency (top Western blots), and IgG signal as a loading control. RNA enrichment was normalized by input total RNA and results were shown as relative RNA enrichment by WT or C512A/C516A (- cisplatin, 100%). Mean ± SD (n = 3) (bottom graphs).