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. 2000 Aug 8;97(17):9618–9623. doi: 10.1073/pnas.160181697

Figure 2.

Figure 2

Stimulation of FL-SMN expression by Htra2-β1 in (A) human HEK293 cells and (B) murine NIH 3T3 fibroblasts. (A) Increasing amounts of Htra2-β1 (0–3 μg) were transiently cotransfected with 1 μg of the pSMN2 minigene in HEK293 cells. pSMN2 recapitulated the splicing pattern of endogenous SMN2, with a ratio of 26.5% (27.7 ± 7.4%) FL-SMN2 to 73.5% (72.3 ± 7.4%) SMN2Δ7 (lane 1). Transient expression of Htra2-β1 led to abundant accumulation of FL-SMN2 in a concentration-dependent manner (lanes 2–4); FL-SMN2 increases to 54.4% (57.9 ± 4.7%) (lane 3) and 72.0% (71.0 ± 8.2%) or 2.7 fold increase of FL-SMN2 (lane 4). M = 100-bp ladder (Life Technologies). Similar results were obtained with a synthetic SMN hybrid gene pSMN1ex7Cen, which recapitulates the splicing pattern of SMN2 (data not shown). (B) Increasing amounts of Htra2-β1 (0–3 μg) transiently cotransfected with a constant amount (1 μg) of pSMN2 minigene into murine NIH 3T3 fibroblasts resulted in a ratio of 55.8% (53.3 ± 3.4%) FL-SMN2 to 44.2% (46.7 ± 3.4%) SMN2Δ7 (lane 1). Transient expression of Htra2-β1 led to increased levels of FL-SMN2 in a concentration-dependent manner (lanes 2–4); FL-SMN2 increased to 77.6% (75.8 ± 2.2%) (lane 3) and up to 79.5% (84.8 ± 7.9%) or 1.4-fold increase (lane 4). However, compared with human, high concentration of Htra2-β1 in murine cells seems to have an inhibitory effect on the transcription level from the minigene. The ratio of the SMN transcripts was determined by semiquantitative analyses using the One-DScan software (MWG Biotec). As an internal control part of the HPRT gene was amplified in a multiplex PCR.