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. 2020 Apr 7;10:6006. doi: 10.1038/s41598-020-61920-4

Figure 4.

Figure 4

BN-PAGE/SDS-PAGE 2D gel electrophoresis analysis – the α1 subunit and c-Src form a protein complex: Preparation of whole cell lysate with Native-PAGE sample buffer were performed as described for BN-PAGE in the Materials and Methods. The experiments were performed as described for BN-PAGE/SDS-PAGE 2D Gel Electrophoresis in the Materials and Methods. Control and BMH-crosslinked samples of LLC-PK1 cells (50 μg/sample) was treated with or without DTT/SDS (100 mM DTT with 1% SDS, final concentration, respectively). For DTT/SDS treatment, samples were heated at 60 °C for 30 min (for the α1 subunit) or 95 °C for 5 min (for c-Src and cav-1). (a) Control sample without DTT/SDS pretreatment. (b) Control sample with DTT/SDS pretreatment. (c) BMH-crosslinked sample without DTT/SDS pretreatment. (d) BMH-crosslinked sample with DTT/SDS pretreatment. Please note the vertical alignment of the α1 subunit and c-Src in (a) but not in (b), which indicated that both proteins were from the same protein complex. Comparing to control (a,b), disappearance of c-Src in (c) and (d) indicated the crosslinking between the α1 subunit and c-Src. n = 3–4.