Fig 1: Untagged Endogenous RAS Proteins Are Degraded with HaloPROTAC-E in Cells Expressing FLAG-Halo-aHRAS(A) Schematic representation of FLAG-Halo-aHRAS HaloPROTAC L-AdPROM system.(B) A549 FLAG-empty, FLAG-aHRAS, and FLAG-Halo-aHRAS-expressing cells were lysed and subjected to IP with anti-FLAG M2 resin. F.T., post-IP flow-through extract.(C) A549 FLAG-Halo-aHRAS-expressing cells were treated with increasing concentrations of HaloPROTAC-E (0–10 µM) for 24 h.(D) A549 FLAG-Halo-aHRAS-expressing cells were treated with 500 nM HaloPROTAC-E for indicated times (0–48 h).(E) A549 FLAG-Halo-aHRAS-expressing cells were treated with 500 nM HaloPROTAC-E and 20 µM proteasome inhibitor MG132 for 24 h.(F) A549 FLAG-aHRAS and FLAG-Halo-aHRAS-expressing cells were treated with 500 nM HaloPROTAC-E for indicated times (0, 3, 6, and 24 h).For (B–F), extracts and IPs were resolved by SDS-PAGE and transferred on to PVDF membranes, which were subjected to immunoblotting with indicated antibodies.(G–K) Quantification from (F) of relative (G) panRAS normalized to GAPDH protein levels (n = 6 ± SD), (H) BRAF normalized to GAPDH protein levels (n = 3 ± SD), (I) p-Y1068 EGFR normalized to total EGFR protein levels (n = 6 ± SD), (J) p-T202/Y204 ERK1/2 normalized to total ERK1/2 protein levels (n = 6 ± SD), and (K) p-S473 Akt normalized to total Akt protein levels (n = 6 ± SD) in the absence or presence of HaloPROTAC-E (500 nM, 24 h).Statistical analyses were carried out by one-way analysis of variance using Tukey's post-test.