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. 2022 Nov 9;42(4):e112275. doi: 10.15252/embj.2022112275

Figure EV2. Orm2 is degraded in WT strains. Related to Fig 5 .

Figure EV2

  1. In the indicated WT strains, degradation of Lcb1‐GFP, Lcb2‐GFP, Orm2‐GFP, Orm2‐GFP, Sac1‐GFP, Tsc3‐GFP, Tsc10‐GFP, and Ypk1‐GFP was measured by CHX‐chase assay (3 biological replicates; n = 3). Cells were analyzed by SDS–PAGE and immunoblotted with α‐GFP.
  2. Steady‐state levels of Dfm1 and corresponding Dfm1 mutants. Cells were analyzed by SDS–PAGE and immunoblotted with α‐HA.
  3. Serial dilution growth assay was performed on dfm1∆orm1∆ and strains with DFM1, DER1‐SHP, DFM1‐AA, DFM1‐Ax3A, DFM1‐5Ashpmtnt, and empty vector add back (3 biological replicates, 2 technical replicates; n = 3).
  4. Same as (C), except serial dilution growth assay was performed on dfm1∆orm1∆ strains with L1 mutant add back: F58S, L64V, K67E, and TMD2 quad mutant add back: DFM1‐R98L, S99V, S100V, and Q101L. Indicated strains were grown on SC‐Leu plates at room temperature, 30°C, and 37°C, and imaged on Day 2 and Day 7 (3 biological replicates, 2 technical replicates; n = 3).
  5. ERAD mutants do not genetically interact with orm1∆. Indicated strains were spotted fivefold dilutions on SC plates in triplicate, and plates were incubated at room temperature, 30°C, and 37°C (3 biological replicates, 2 technical replicates; n = 3).