Figure 2 | Cell Death & Disease

Figure 2

From: Ubiquitylation of MFHAS1 by the ubiquitin ligase praja2 promotes M1 macrophage polarization by activating JNK and p38 pathways

Figure 2

MFHAS1 forms a complex with praja2. (a) HEK293 cells were transiently transfected with a plasmid expressing His-tagged MFHAS1. After 24 h of transfection, the cells were treated with MG132 (20 μM) for 6 h and were collected. The cell lysates were immunoprecipitated using anti-praja2 or anti-His antibody. (b) HEK293-MFHAS1 cell lysates were used for performing pulldown assays with purified GST or GST-praja2 fusion protein, followed by immunoblotting with anti-GST and anti-HA antibodies. (c) HEK293-MFHAS1 cells were subjected to double immunostaining with monoclonal anti-HA and polyclonal anti-praja2 antibodies. The images were collected and were analyzed using a confocal microscope. Magnification of selected areas is shown (insets). Scale bar, 10 μm. (d) Schematic representation of MFHAS1 constructs used in this study. (e) HEK293 cells were transiently co-transfected with plasmids expressing Flag-praja2 and HA-tagged ΔLRR-MFHAS1 (Δ64-364), ΔROC-MFHAS1 (Δ403-649), ΔCOR-MFHAS1 (Δ650-980) or ΔLRR-ROC-MFHAS1 (Δ64-364,403-649). After 24 h of transfection, the cells were treated with MG132 (20 μM) for 6 h and were lysed for performing immunoprecipitation with anti-Flag antibody

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