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. 2022 Sep 21;54(9):1549–1562. doi: 10.1038/s12276-022-00849-2

Fig. 6. PRKD3 suppressed lysosomal CLU degradation via its kinase activity.

Fig. 6

a, b Reciprocal immunoprecipitation showing the interaction between PRKD3 and CLU. c Representative images from the immunofluorescence assay showing the colocalization of PRKD3 and CLU using specific antibodies. PRKD3 (green), CLU (red) and DAPI (blue). The scale bar indicates 50 μm. d Representative western blot showing that the protein level of CLU was not significantly different among the three groups. e Representative western blot showing that treatment of LF cells with siPRKD3 decreased CLU protein levels compared with the expression level of WT cells. f RT‒PCR findings showing that there were no marked differences in CLU mRNA levels in the siPRKD3 LF cells compared with the WT cells. g, h Western blots showing that treatment with the lysosomal degradation suppressor CQ but not the proteasomal degradation suppressor MG132 elevated the protein levels of CLU in the siPRKD3-treated LF cells. i Representative confocal images from the immunofluorescence assay showing a significant increase in CLU-LAMP1 colocalization foci in the siPRKD3-treated LF cells compared with the WT cells. Representative CLU-LAMP1 colocalization foci are shown by red arrows. Scale bar: 5 µm. j Representative western blots showing a decrease in CLU protein levels in LF cells after treatment with CRT0066101 compared with the cells treated with PBS. k RT‒PCR results showing that there was no marked difference in CLU and PRKD3 mRNA levels after treatment with the PRKD3 kinase activity inhibitor CRT0066101 relative to treatment with PBS. l Representative confocal images from immunofluorescence assays showing a significant increase in CLU-LAMP1 colocalization foci in LF cells upon treatment with CRT0066101 compared with treatment with PBS. Representative CLU-LAMP1 colocalization foci are shown by the red arrow. Scale bar: 5 µm. m Representative western blots showing the effects of overexpression of PRKD3 on the protein levels of ECM proteins in LF cells with or without TGF-β1 treatment. n Representative western blots showing the effects of siPRKD3 on the protein levels of ECM proteins in LF cells with or without TGF-β1 treatment. o Representative western blots showing the effects of CRT0066101 on the protein levels of ECM proteins in LF cells with or without TGF-β1 treatment.