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CSFV infection causes the <t>PERK</t> pathway-mediated autophagy to promote viral replication in cultured PK-15 cells . (a–d) PK-15 cells cultured in 12-well plates were respectively pretreated with 1 μM CCT, 1 μM GSK, 1 μM SAL or equal amount of DMSO for 1 h, and then subject to CSFV infection (1 MOI) for 1.5 h. The cells were further cultured in the presence of the chemicals for 24 h, and then collected for qRT-PCR (a), Western blot (b and c) and TCID 50 assays (d), respectively. For qRT-PCR (a), relative quantification of ATG5, Beclin1, ATF4, CHOP and NS5B genes were assessed using the 2 −ΔΔCT method and normalized to GAPDH . Two-way ANOVA tests: *, P < 0.05; **, P < 0.01. For Western blot (b), samples were prepared as described in the “materials and methods” for Western blot analyses using specific antibody against <t>p-PERK,</t> total PERK, p-eIF2α, total eIF2α, ATG5, p62, LC3B, N pro and tubulin. Grayscale value of the bands were analyzed with ImageJ software, and generations of images for protein quantification were performed with GraphPad Prism 6 software. Two-way ANOVA tests: *, P < 0.05; **, P < 0.01; ***, P < 0.001;****, P < 0.0001 (c). For virus titration (d), the cells and supernatants of each treatment were collected for TCID 50 assays through an indirect immunofluorescence assay using mouse anti-CSFV E2 antibody. Virus titers were expressed as Log 10 (TCID 50 /mL). Two-way ANOVA tests: *, P < 0.05. (e–h) PK-15 cells cultured in 12-well plates were respectively transfected with 10 nM SiPERK or 10 nM SiNC for 4 ~ 6 h, and further incubated at 37°C for 12 h prior to a 1.5 h of incubation with 1 MOI of CSFV. The cells were further cultured for 24 h, and then collected for detection of relative mRNA expression of ATG5, ATG12, Beclin1 and NS5B genes by qRT-PCR (e), and determination of protein expression of p-PERK, total PERK, p-eIF2α, total eIF2α, ATG5, p62, LC3B, N pro , and tubulin through Western blot analysis (f and g), as well as measurement of virus titers with TCID 50 assays (h). All the data shown are expressed as mean ± SD values of two independent experiments, and are analyzed by two-way ANOVA tests: *, P < 0.05; **, P < 0.01; ***, P < 0.001;****, P < 0.0001

Phosphor P Perk, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 96/100, based on 934 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/phosphor+p+perk/Phospho-PERK+(Thr980)+Rabbit+mAb/pmc07781608-53-10-13
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phosphor p perk - by Bioz Stars, 2026-09

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1) Product Images from "Classical swine fever virus employs the PERK- and IRE1-dependent autophagy for viral replication in cultured cells."

Article Title: Classical swine fever virus employs the PERK- and IRE1-dependent autophagy for viral replication in cultured cells.

Journal: Virulence

doi: 10.1080/21505594.2020.1845040

CSFV infection causes the PERK pathway-mediated autophagy to promote viral replication in cultured PK-15 cells . (a–d) PK-15 cells cultured in 12-well plates were respectively pretreated with 1 μM CCT, 1 μM GSK, 1 μM SAL or equal amount of DMSO for 1 h, and then subject to CSFV infection (1 MOI) for 1.5 h. The cells were further cultured in the presence of the chemicals for 24 h, and then collected for qRT-PCR (a), Western blot (b and c) and TCID 50 assays (d), respectively. For qRT-PCR (a), relative quantification of ATG5, Beclin1, ATF4, CHOP and NS5B genes were assessed using the 2 −ΔΔCT method and normalized to GAPDH . Two-way ANOVA tests: *, P < 0.05; **, P < 0.01. For Western blot (b), samples were prepared as described in the “materials and methods” for Western blot analyses using specific antibody against p-PERK, total PERK, p-eIF2α, total eIF2α, ATG5, p62, LC3B, N pro and tubulin. Grayscale value of the bands were analyzed with ImageJ software, and generations of images for protein quantification were performed with GraphPad Prism 6 software. Two-way ANOVA tests: *, P < 0.05; **, P < 0.01; ***, P < 0.001;****, P < 0.0001 (c). For virus titration (d), the cells and supernatants of each treatment were collected for TCID 50 assays through an indirect immunofluorescence assay using mouse anti-CSFV E2 antibody. Virus titers were expressed as Log 10 (TCID 50 /mL). Two-way ANOVA tests: *, P < 0.05. (e–h) PK-15 cells cultured in 12-well plates were respectively transfected with 10 nM SiPERK or 10 nM SiNC for 4 ~ 6 h, and further incubated at 37°C for 12 h prior to a 1.5 h of incubation with 1 MOI of CSFV. The cells were further cultured for 24 h, and then collected for detection of relative mRNA expression of ATG5, ATG12, Beclin1 and NS5B genes by qRT-PCR (e), and determination of protein expression of p-PERK, total PERK, p-eIF2α, total eIF2α, ATG5, p62, LC3B, N pro , and tubulin through Western blot analysis (f and g), as well as measurement of virus titers with TCID 50 assays (h). All the data shown are expressed as mean ± SD values of two independent experiments, and are analyzed by two-way ANOVA tests: *, P < 0.05; **, P < 0.01; ***, P < 0.001;****, P < 0.0001

Figure Legend Snippet: CSFV infection causes the PERK pathway-mediated autophagy to promote viral replication in cultured PK-15 cells . (a–d) PK-15 cells cultured in 12-well plates were respectively pretreated with 1 μM CCT, 1 μM GSK, 1 μM SAL or equal amount of DMSO for 1 h, and then subject to CSFV infection (1 MOI) for 1.5 h. The cells were further cultured in the presence of the chemicals for 24 h, and then collected for qRT-PCR (a), Western blot (b and c) and TCID 50 assays (d), respectively. For qRT-PCR (a), relative quantification of ATG5, Beclin1, ATF4, CHOP and NS5B genes were assessed using the 2 −ΔΔCT method and normalized to GAPDH . Two-way ANOVA tests: *, P < 0.05; **, P < 0.01. For Western blot (b), samples were prepared as described in the “materials and methods” for Western blot analyses using specific antibody against p-PERK, total PERK, p-eIF2α, total eIF2α, ATG5, p62, LC3B, N pro and tubulin. Grayscale value of the bands were analyzed with ImageJ software, and generations of images for protein quantification were performed with GraphPad Prism 6 software. Two-way ANOVA tests: *, P < 0.05; **, P < 0.01; ***, P < 0.001;****, P < 0.0001 (c). For virus titration (d), the cells and supernatants of each treatment were collected for TCID 50 assays through an indirect immunofluorescence assay using mouse anti-CSFV E2 antibody. Virus titers were expressed as Log 10 (TCID 50 /mL). Two-way ANOVA tests: *, P < 0.05. (e–h) PK-15 cells cultured in 12-well plates were respectively transfected with 10 nM SiPERK or 10 nM SiNC for 4 ~ 6 h, and further incubated at 37°C for 12 h prior to a 1.5 h of incubation with 1 MOI of CSFV. The cells were further cultured for 24 h, and then collected for detection of relative mRNA expression of ATG5, ATG12, Beclin1 and NS5B genes by qRT-PCR (e), and determination of protein expression of p-PERK, total PERK, p-eIF2α, total eIF2α, ATG5, p62, LC3B, N pro , and tubulin through Western blot analysis (f and g), as well as measurement of virus titers with TCID 50 assays (h). All the data shown are expressed as mean ± SD values of two independent experiments, and are analyzed by two-way ANOVA tests: *, P < 0.05; **, P < 0.01; ***, P < 0.001;****, P < 0.0001

Techniques Used: Infection, Cell Culture, Quantitative RT-PCR, Western Blot, Quantitative Proteomics, Software, Virus, Titration, Immunofluorescence, Transfection, Incubation, Expressing

CSFV infection causes the PERK pathway-mediated autophagy to promote viral replication in cultured 3D4/2 cells . (a–d) 3D4/2 cells were treated with PERK pathway regulators and CSFV infection as described in the legend of , and then collected for detection of relative mRNA expression of ATG5, Beclin1, ATF4, CHOP and NS5B genes by qRT-PCR (a), and determination of protein expression of p-PERK, total PERK, p-eIF2α, total eIF2α, ATG5, p62, LC3B, N pro , and tubulin through Western blot analysis (b and c), as well as measurement of virus titers with TCID 50 assays (d). (e–h) 3D4/2 cells were treated with SiPERK and CSFV infection as described in the legend of , and then collected for detection of relative mRNA expression of ATG5, ATG12, Beclin1 and NS5B genes by qRT-PCR (e), and determination of protein expression of p-PERK, total PERK, p-eIF2α, total eIF2α, ATG5, p62, LC3B, N pro and tubulin through Western blot analysis (f and g), as well as measurement of virus titers with TCID 50 assays (h). All the data shown are expressed as mean ± SD values of two independent experiments. Two-way ANOVA tests: *, P < 0.05; **, P < 0.01; ***, P < 0.001;****, P < 0.0001

Figure Legend Snippet: CSFV infection causes the PERK pathway-mediated autophagy to promote viral replication in cultured 3D4/2 cells . (a–d) 3D4/2 cells were treated with PERK pathway regulators and CSFV infection as described in the legend of , and then collected for detection of relative mRNA expression of ATG5, Beclin1, ATF4, CHOP and NS5B genes by qRT-PCR (a), and determination of protein expression of p-PERK, total PERK, p-eIF2α, total eIF2α, ATG5, p62, LC3B, N pro , and tubulin through Western blot analysis (b and c), as well as measurement of virus titers with TCID 50 assays (d). (e–h) 3D4/2 cells were treated with SiPERK and CSFV infection as described in the legend of , and then collected for detection of relative mRNA expression of ATG5, ATG12, Beclin1 and NS5B genes by qRT-PCR (e), and determination of protein expression of p-PERK, total PERK, p-eIF2α, total eIF2α, ATG5, p62, LC3B, N pro and tubulin through Western blot analysis (f and g), as well as measurement of virus titers with TCID 50 assays (h). All the data shown are expressed as mean ± SD values of two independent experiments. Two-way ANOVA tests: *, P < 0.05; **, P < 0.01; ***, P < 0.001;****, P < 0.0001

Techniques Used: Infection, Cell Culture, Expressing, Quantitative RT-PCR, Western Blot, Virus

Proposed model for CSFV-induced autophagy via cellular PERK and IRE1 pathway . CSFV infection induces ERS and then triggers autophagy through the PERK-eIF2α-ATF4-CHOP and the IRE1/GRP78 pathway, thus facilitating viral replication in cultured cells

Figure Legend Snippet: Proposed model for CSFV-induced autophagy via cellular PERK and IRE1 pathway . CSFV infection induces ERS and then triggers autophagy through the PERK-eIF2α-ATF4-CHOP and the IRE1/GRP78 pathway, thus facilitating viral replication in cultured cells

Techniques Used: Infection, Cell Culture

Infection:

Article Title: Classical swine fever virus employs the PERK- and IRE1-dependent autophagy for viral replication in cultured cells.
Article Snippet: The primary antibodies used in our study were specific for phosphor (p)-PERK (Thr980) (Cell Signaling, 3179), total PERK (Cell Signaling, 3192), p-eIF2α (S51) (Bioworld, BS4787), total eIF2α (Bioworld, BS3651), GRP78 (Santa Cruz, sc-13968), ATG5 (Novus Biologicals, NB110-53818), SQSTM1/p62 (Cell Signaling, 39749), LC3B (Cell Signaling, 2775), CSFV-E2 (MEDIAN/JBT, 9011), Tubulin (Beyotime, AT819), and CSFV-Npro (kindly donated by professor Xinglong Yu, Hunan Agricultural University, China).

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