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. 2019 Aug 5;20(15):3817. doi: 10.3390/ijms20153817

Figure 5.

Figure 5

Evaluation of the interaction between UmHal3 variants with ScPpz1 and UmPpz1. Glutathione beads containing 4 μg of the GST-tagged UmPpz1 (panel A) or 8 μg of ScPpz1 and ScPpz1-Cter (panel B) were immobilized on glutathione beads and mixed with extracts from IM021 (ppz1 hal3) cells expressing 3HA-tagged ScHal3, UmHal3, UmHal3_PD (PD) or UmHal3_PD_ScCter (PD_ScCt) from the pWS93 plasmid. Beads were washed, resuspended in 100 μl of 2x sample buffer and processed for SDS-PAGE (6% gels for UmHal3 and 10% for PD and PD_ScCter). Typically, 25 μl of the samples was used, except in lanes marked with an asterisk, where 50 μl was employed. Proteins were immunoblotted using anti-HA antibodies as described in Methods. Ponceau staining of the membranes is shown to evaluate for correct loading and transfer (ScPpz1-Cter was not seen in the left blot in panel B because due to its relatively low molecular mass run with the front of the 6% gel).