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

Figure 2.

Figure 2

Validation of crosslinking and capillary immunoblotting analysis of LLC-PK1 cells: (a) LLC-PK1 cells were processed with or without BMH crosslinking as described in the Materials and Methods. Whole cell lysates were prepared with Nonidet P-40 lysis buffer, and then processed for traditional immunoblotting analysis under denature condition (heated at 60 °C for 30 min for the α1 subunit, and 95 °C for 5 min for c-Src and cav-1 detection) with SDS-PAGE. 60 µg of total protein per sample was used. After transferring, PVDF membranes were cut below the molecular weight of monomers, i.e., below 75 kDa for the α1 subunit, below 50 kDa for total c-Src, and below 20 kDa for cav-1. ECL was used to develop blots. n = 2–3. (b) After crosslinking, whole cell lysates of LLC-PK1 cells were prepared with 1x Native-PAGE sample buffer (with 2% DDM). The lysates (3 μg protein/sample) were treated without or with DTT (final concentration 40 mM) plus SDS (final concentration 1%), and then processed side-by-side on the same 66–440 kDa separation module and immunoblotted with detection module (Wes system, ProteinSimple) for capillary immunoblotting analysis against the α1 subunit, total c-Src, and cav-1. 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). Lane 1, control with mocking crosslinking process; Lane 2, BMH-crosslinked; and Lane 3, DTME-crosslinked. n = 5.