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. 2020 May 20;15(5):e0233502. doi: 10.1371/journal.pone.0233502

Fig 3. ERp29 interacts with Sec24D and Proinsulin.

Fig 3

Min-6 cells were lysed under non-denaturing conditions, and 500 μg of total lysate protein was subject to immunoprecipitation with either anti-ERp29 (A, D), anti-Sec24D (B), or anti-(Pro)insulin (C). Precipitated proteins were resolved by SDS-PAGE and immunoblots were probed for Sec24D, ERp29, or (Pro)insulin as indicated. The Min-6 lysate lanes were loaded with 50 μg (10%) of the input lysate. These data are representative of n = 4 independent experiments. The co-precipitated Sec24D band is denoted with asterisk in panel (A). (E) Fifty μg of Min-6 whole cell lysate prepared under non-denaturing conditions was subject to immunoprecipitation under the following conditions: No antibody control (n = 12), anti-V5 (non-specific control, n = 11), anti-ERp29 (n = 14), anti-C-peptide from Novus (n = 14), anti-(Pro)Insulin from Sigma (n = 12), and anti-(Pro)Insulin from Cell Signaling Technologies (CST, n = 11). Precipitated Proinsulin was quantified by a rat/mouse Proinsulin ELISA from Mercodia as specified in the materials and methods. Anti-V5 control vs anti-ERp29, p = 0.0262; anti-V5 control vs anti-C-peptide, p = 0.0048; anti-V5 control vs Sigma antibody, p = <0.0001; anti-V5 control vs CST antibody, p = <0.0001.