In our analysis, the total APP expression of both mature (upper band) and immature (lower band) forms was significantly elevated approximately 2 to 7-fold in MCF10A (M-II, -III, and -IV) cells compared to M-I cells.
The glucose-induced doublet (panel E, lanes 2, 3, 4, 6 and 8) corresponds to ubiquitylated (higher band) and non-ubiquitylated (lower band) forms of Rod1, because mutation of its ubiquitylation sites (using a plasmid encoding Rod1-KR) abolishes the migration shift (panel F, lane 6), as described previously (Becuwe et al., 2012b ).
Quantification of SCD-1 activity was determined based on the intensity of 14C oleate (lower band) formed relative to the 14C stearate (upper band).
On Westerns, this transition could be seen by loss of the upper hyper-phosphorylated band, whereas the lower band represents hypophosphorylated forms of Rb.
The data of a representative transformant are shown in Figure 2. The majority of the cells (88%) were flagellated and motile (Panel A) and strong signals corresponding to the unphosphorylated (lower band) and phosphorylated (upper band) forms of the RSP3-HA protein were detected in a Western blot using the anti-HA antibody (Panel B).
However, p21 mutants, like wild-type p21, inhibit Cdk, as is evident by diminished in vitro Cdk activity against the substrate histone H1 or by an endogeneous cellular decrease in hyperphosphorylated form (upper band) and a concomitant increase in hypophosphorylated form (lower band) of the Rb substrate.
Wild-type feline Tetherin/BST-2 was detected as triplet bands, while N79A and N119A mutants and N79A/N119A mutant showed double and single band(s), respectively (Figure 4), indicating that the upper, middle, and lower bands of triplet forms corresponded to multiple-, single-, and non-glycosylated forms, respectively.
The Leu-150 form (lower band) contains Cys-155 and thus differs from commonly produced ΔN-HtrA1 that lacks this residue.
Following reducing conditions, three major bands were observed via SDS PAGE, corresponding to fragments starting at Val-111 (top band), Gln-131 (middle band), and Leu-150 (lower band), in agreement with the forms observed in vivo.
The lower band represents the ER precursor form that lacks complex sugar modifications, while the upper band represents the mature form of the receptor [16].
Therefore, the upper band represents most likely the unprocessed precursor, whereas the lower band corresponds to the mature form.
