Fig. 4

Alterations in CAMKK2, CAMK4, TF, and TFRC-associated MPCs. a-d: Immunoblots showing CAMKK2 (a-b) and CAMK4-associated MPCs (c-d) in the wild-type (WT) and CAMKK2^−/− HEK293 cells. The red circles represent the vertical alignment of ~ 146 kDa CAMKK2 and CAMK4-associated MPCs which indicates the possibility that these proteins occupied the same MPCs. The red arrow in D indicates that CAMK4-associated MPCs shifted to a lower molecular weight complex in the absence of CAMKK2 which is probably due to the loss of an interacting partner. The top Coomassie-stained gel slice is showing the separation of native-PAGE markers in the first-dimension along with the separation of total cellular proteins in the next lane. The MPCs presented in the A-D were separated in the same first-dimension BN-PAGE, therefore, relative migration of the MPCs are comparable in the second-dimension SDS-PAGE. The nonspecific (ns) band in the anti-CAMKK2 immunoblot is persistence in the CAMKK2-deficient cells indicating non-specific binding of the antibody. e-m: Immunoblots showing alterations in the endogenously expressed TF and native CAMKK2-associated MPCs in the wild-type, CAMKK2^−/−, CAMK4^−/− and DKO HEK293 cells. The cells were transiently transfected with TF for 6 h and the protein lysates were subjected to BN-PAGE/SDS-PAGE analysis. Red rectangles (E-M) indicate the vertical alignment of CAMKK2 and TF-associated > 1200 kDa MPCs, which is dramatically reduced in DKO cells (l). Blue rectangles (e, h, j, and l) indicate multiple TF-associated MPCs in the range of 480–1200 kDa which exhibited considerable difference in CAMKK2-deficient cells. Red circles (e, h, j, and l) indicate a ~ 146 kDa TF associated MPC that is differentially shifted to higher molecular weight complexes in CAMKK2^−/−, CAMK4^−/− and DKO cells. Blue circles (f, i, k, and m) indicate CAMKK2-associated ~ 146 kDa MPCs vertically aligned with the TF-associated MPCs. The loss of CAMKK2 and/or CAMK4 caused a considerable shift in the ~ 146 kDa TF-associated MPCs which indicates potential functional dependency between these proteins. n-q: Immunoblots showing alterations in the CAMKK2- and TFRC-associated MPCs in the serum-starved wild-type and CAMKK2^−/− HEK293 cells treated with or without 25 μg/ml TF for 30 mins. The immunoblots presented here were first immunoblotted using anti-TFRC antibody and subsequently immunoblotted using anti-CAMKK2 antibody to detect co-migration of TFRC and CAMKK2 associated protein complexes respectively. The anti-TFRC immunoblots were presented in Fig. 5 to exhibit co-migration of TFRC with TF-associated proteins. Native TFRC is an 84kda protein but a 100 kDa TFRC and 135 kDa RFP-TFRC is visible in the immunoblots. In addition, > 135 kDa TFRCs were also present. All high molecular fraction of TFRC may be due to PTMs because the denatured (50 °C) and reducing (100 mM DTT) second-dimension SDS-PAGE would disrupt any potential dimers or oligomers formation due to intermolecular disulfide-bridge. Green and blue rectangles indicate the vertical alignment of > 1200 kDa TFRC/RFP-TFRC and CAMKK2-associated MPCs. The relative amount of > 1200 kDa TFRC-associated MPCs was comparatively reduced in CAMKK2^−/− cells indicating potential functional implication and interdependency. Red arrows indicate a relative shift of > 480 kDa TFRC-associated MPCs in untreated and TF-treated wild-type and CAMKK2^−/− cells. Red rectangles indicate CAMKK2-associated 146 kDa MPCs. “-” means “without TF treatment”; “+” means “with TF treatment”; “=” means “two bands of the same protein”.