A. 50. YB1. 47. 35. actin. 10nm. 25nm. 50nm. 75nm. 100 nm. Control siRNA.
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A)Cells were seeded at 10-15,000 cells per ml (per well) in triplicate in 24 well plates. After 24 hours cells were transfected with a pool of 3 YB1 targeted siRNAs from 10 to 100nM concentrations (Dharmacon On-TARGETplus pools of YBX1 NM_004559 , LQ-010213-00-0005), using 1ul of Lipofectamine RNAiMAX (Invitrogen) per well. Cells were harvested after 48 hours and proteins were separated on 10% SDS PAG and immunoblotted using the YB1 antibody (obtained from ABCAM, 12148). Control cells were transfected with 75nM siRNA non-targeting control #3 (D-001810-03-20) .Bands approximately 50, 47 and 35kDa are reduced in the presence of the siRNAs suggesting that these correspond to isoforms of YB1 proteins.
Cold shock domain
B) Impact of individual siRNAs on knockdown of YB1 in HeLa cells. The siRNAs targeting YB1 (see above) were used individually to assess the impact of each upon YB1 expression (i). The regions of the four known YB1 isoforms (obtained from www.ensembl.org) targeted by siRNAs are illustrated by black bars (iii). Cells were transfected as described above, with 25nM of the single siRNAs. After 72 hours the cells were harvested and Western blots carried out. With reference to the full-length YB1 1 001 cDNA sequence, the binding sites of the different siRNAs are as follows: siRNA#08 372-391nt; siRNA#07 1033-1052nt & siRNA#06 1139-1159nt. Individual siRNAs did not affect the large (50kDa) and small (35kDa) bands to the same extent, suggesting that one or more different YB1 isoforms are targeted in each case. The 50kDa band is reduced in the presence of each siRNA, confirming that it corresponds to the full-length 324aa transcript with predicted molecular weight of 36kDa. The 35kDa band showed a significant knockdown at this concentration of siRNA with siRNA#07, and a small knock down with siRNA#06. This would suggest that this band does not correspond to YB1 isoform 004 (216aa, predicted weight 23.6kDa), which lacks the corresponding target site. Alternatively, it is unlikely that the smaller band corresponds to known cleavage products (Sorokin et al 2005) since these migrate at 32kDa and 22kDa respectively and would be expected to be reduced by a similar extent by siRNA knockdown.
This leaves the possibility that the smaller band corresponds to one of the following isoforms: (i) full length YB1-001 protein migrating at a size close to that predicted by its molecular weight, and therefore unmodified; (ii) YB1-002, 374aa, or; (iii) YB1-201, 314aa, predicted molecular weight 34kDa.