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. 2022 Oct 5;96(20):e00549-22. doi: 10.1128/jvi.00549-22

FIG 7.

FIG 7

SYNJ2BP reduces ER stress induced by EIAV or HIV Env. (A) EIAV Env can induce increases in protein expression levels of the ER stress marker Grp78 in a dose-dependent manner. HEK293T cells were transfected with either pcDNA3.1-Env (EIAV) plasmids in two doses or an empty plasmid, and Tu or Tg (2 μg/mL) was used as a positive control for ER stress activation. Cell lysates were analyzed by Western blotting to detect the intensity of the Grp78 protein band at 48 hpt. The results of the densitometry analysis to quantify the ratio of Grp78 to β-actin are shown at the bottom (lane 1 set as 1.0). This experiment was performed three times. (B) EIAV Env can induce increases in mRNA levels of the ER stress marker Grp78. HEK293T cells were transfected with either pcDNA3.1-Env (EIAV) plasmids or an empty plasmid. Total RNA was isolated, and the transcriptional levels of Grp78 were measured by qPCR at 48 hpt. Fold change values were calculated according to the 2–ΔΔCT method using β-actin as an internal reference gene. The data represent the means ± the SEM from three independent experiments (**, P < 0.01). (C) EIAV Env was degraded through the ERAD pathway. KIF restores EIAV Env expression in HEK293T cells. HEK293T cells were transfected with plasmid pcDNA3.1-Env (EIAV) in treatment with KIF (100 μM) or without KIF. Cells were lysed, and EIAV Env expression levels were determined by Western blotting. The results of the densitometry analysis to quantify the ratio of Env to β-actin are shown at the bottom (lane 1 set as 1.0). This experiment was performed three times. (D) SYNJ2BP overexpression can reduce high expression levels of Grp78 induced by EIAV Env. HEK293T cells were cotransfected with either pcDNA3.1-Flag-SYNJ2BP plasmids or an empty plasmid, and pcDNA3.1-Env (EIAV) or an empty plasmid, and Tu (2 μg/mL) was used as a positive control for ER stress activation. Cell lysates were analyzed using Western blotting to detect the intensity of the Grp78 or SYNJ2BP-Flag protein bands at 48 hpt. The results of the densitometry analysis to quantify the ratio of Grp78 to β-actin are shown at the bottom (lane 1 set as 1.0). This experiment was performed three times. (E) SYNJ2BP overexpression can reduce high expression levels of Grp78 induced by HIV Env. HEK293T cells were cotransfected with either pcDNA3.1-Flag-SYNJ2BP plasmids or an empty plasmid, and pcDNA3.1-Env-HA (HIV-1) or an empty plasmid, and Tu (2 μg/mL) was used as a positive control for ER stress activation. Cell lysates were analyzed using Western blotting to detect the intensity of the Grp78 or SYNJ2BP-Flag protein bands at 48 hpt. The results of the densitometry analysis to quantify the ratio of Grp78 to β-actin are shown at the bottom (lane 1 set as 1.0). This experiment was performed three times.