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. 2021 Dec 13;25(1):103605. doi: 10.1016/j.isci.2021.103605

Figure 2.

Figure 2

IL-32 is localized to the mitochondria and interacts with components of the mitochondrial respiratory chain

(A) CO-IP was performed by pull-down of endogenous IL-32 in INA-6, JJN-3, and H929 cells. Representative immunoblots of ATP5D, NDUFA12, and IL-32 are shown. The vertical lines in the IL-32 lanes are to indicate that to improve visualization contrast/brightness were adjusted differently for the total cell lysate (2 lanes to the left) and for the IP samples (4 lanes to the right).

(B) Representative immunoblot of IL-32 in the mitochondrial and cytosolic fraction of JJN-3 cells cultured in normoxia (20% oxygen) and hypoxia (2% oxygen).

(C) Representative confocal image of hypoxic JJN-3 cells stained for IL-32 (magenta, Alexa 647), mitochondria (TOMM20, green, Alexa 488), and nucleus (blue, Hoechst). Imaging was performed with a Leica SP9, using a 63 × 1.4 (oil) objective and LAS X software and deconvoluted using Huygens. Scale bar: 5μM. Arrows indicate areas of colocalization of TOMM20 and IL-32. Correlation rate (CR, in %) is the mean ± SD calculated from N = 4 images analyzed in Leica Application Suite X.