Fig. 5

Alteration of TF and TFRC-associated MPCs during trafficking in the wild-type, CAMKK2^−/−, CAMK4^−/−, and DKO HEK293 cells. (ab+ef & ij + mn): Immunoblots showing alterations of TFRC-associated MPCs in serum-starved wild-type (a-b), CAMKK2^−/− (e-f), CAMK4^−/−(i-j) and DKO (m-n) HEK293 cells transiently-transfected with RFP-TFRC for 6 h and treated with or without 25 μg/ml TF for 30 mins. The immunoblots representing different knockout cell clones are color-coded. The MPCs in each second-dimension-derived immunoblot were separated together in the same first-dimension BN-PAGE. The immunoblots are aligned to show relative migration of the protein complexes. Red rectangles (ab+ef & ij + mn) indicate the vertical alignment of > 1200 kDa TFRC-associated MPCs, which is dramatically reduced in CAMKK2^−/−, CAMK4^−/−, and DKO cells. Red arrows indicate the relative position of native ~ 480–720 kDa TFRC-associated MPCs. Blue arrows showing the relative shift in native ~ 480–720 kDa TFRC-MPCs in TF-treated CAMKK2^−/−, CAMK4^−/−, and DKO cells. The Coomassie-stained native page markers provided at the top of each group of immunoblots (AB&EF/ IJ&MN) indicate proteins separated in the same first dimension BN-PAGE. Tx: treatment with TF. (cd + gh & kl + op): The anti-TFRC immunoblots from ab+ef & ij + mn were false colored and overlaid with corresponding anti-TF immunoblots to show co-migration of the TF/TFRC-associated MPCs during trafficking in the TF-treated/untreated CAMKK2 and/or CAMK4-deficient cells. The MPCs within each color-coded (grey/orange/blue/green) panels were separated in the same first-dimension BN-PAGE; therefore, their relative shift in position is comparable. Vertical alignment indicates potential association between the same MPCs. White arrows indicate a relative shift in the TF and TFRC-associated MPCs. The anti-TFRC immunoblots presented in Fig. 5a, b, e, and f were also presented in Fig. 4 n-q after immunoblotting with anti-CAMKK2 antibody. “No TF”: without TF treatment; “+TF”: treatment with TF. White and red arrows showing the relative shift in native ~ 480–720 kDa TFRC-MPCs in TF-treated CAMKK2^−/−, CAMK4^−/−, and DKO cells. White dotted rectangles indicate the vertical alignment of > 1200 kDa TF/TFRC-associated MPCs, which is dramatically reduced in CAMKK2^−/−, CAMK4^−/−, and DKO cells.