gfp (Santa Cruz Biotechnology)

Santa Cruz Biotechnology is a verified supplier

About

News

Press Release

Team

Advisors

Partners

Contact

Bioz Stars

Bioz vStars

Buy from Supplier

Structured Review

Santa Cruz Biotechnology gfp

Figure 2. ER stress promotes the aggregation of mtHTT via IRE1 activation. (A) ER stress enhances mtHTT aggregation. SH-SY5Y cells were co-transfected with <t>pHTTex120Q-GFP</t> and either pcDNA or pIRE1, and then exposed to DMSO, 1 mm thapsigargin (Tg) or 2 mg/ml tunicamycin (Tuni.) for the indicated times. Cells were then observed under ﬂuorescence microscope (left panel) or cell lysates were examined with western blot analysis using the indi- <t>cated</t> <t>antibodies</t> (right panel). Bars represent mean values+SD from at least three independent experiments. P-values were calculated using t-test and were versus control (∗P , 0.05; ∗∗P , 0.01; ∗∗∗P , 0.001). (B) Down-regulation of IRE1 reduces ER stress-induced mtHTT aggregation. SH-SY5Y/pSuper-Neo (SH/Neo) and SH-SY5Y/pSuper-shIRE1 (SH/shIRE1 #5 and #8) cells were transfected with pHTTex120Q-GFP and incubated with DMSO or 1 mm thapsi- gargin (Tg) for 24 h. Percentages of mtHTT aggregation were determined under ﬂuorescence microscope as in (A) (left panel). Cell extracts were prepared and examined by western blot analysis using the indicated antibodies (right panel). (C) Inhibitory effect of dominant-negative IRE1 on ER stress-induced mtHTT aggregation. After co-transfection with pHTTex120Q-GFP and either pcDNA or each dominant-negative mutant of ER sensor proteins (IRE1 DN, ATF6 DN and PERK DN), SH-SY5Y cells were left untreated or exposed to 1 mm thapsigargin (Tg). The aggregation of mtHTT was examined under ﬂuorescence microscope (left panel) and cell extracts were analyzed with western blotting using anti-HA, anti-GRP78 and anti-b-actin antibodies (right panel). Arrowheads indicate the expression of each construct.

Gfp, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 96/100, based on 7857 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/trapz+function/pm21954231-261-45-46?v=Santa+Cruz+Biotechnology
Average 96 stars, based on 7857 article reviews

gfp - by Bioz Stars, 2026-08

96/100 stars

Images

1) Product Images from "IRE1 plays an essential role in ER stress-mediated aggregation of mutant huntingtin via the inhibition of autophagy flux."

Article Title: IRE1 plays an essential role in ER stress-mediated aggregation of mutant huntingtin via the inhibition of autophagy flux.

Journal: Human molecular genetics

doi: 10.1093/hmg/ddr445

Figure Legend Snippet: Figure 2. ER stress promotes the aggregation of mtHTT via IRE1 activation. (A) ER stress enhances mtHTT aggregation. SH-SY5Y cells were co-transfected with pHTTex120Q-GFP and either pcDNA or pIRE1, and then exposed to DMSO, 1 mm thapsigargin (Tg) or 2 mg/ml tunicamycin (Tuni.) for the indicated times. Cells were then observed under ﬂuorescence microscope (left panel) or cell lysates were examined with western blot analysis using the indi- cated antibodies (right panel). Bars represent mean values+SD from at least three independent experiments. P-values were calculated using t-test and were versus control (∗P , 0.05; ∗∗P , 0.01; ∗∗∗P , 0.001). (B) Down-regulation of IRE1 reduces ER stress-induced mtHTT aggregation. SH-SY5Y/pSuper-Neo (SH/Neo) and SH-SY5Y/pSuper-shIRE1 (SH/shIRE1 #5 and #8) cells were transfected with pHTTex120Q-GFP and incubated with DMSO or 1 mm thapsi- gargin (Tg) for 24 h. Percentages of mtHTT aggregation were determined under ﬂuorescence microscope as in (A) (left panel). Cell extracts were prepared and examined by western blot analysis using the indicated antibodies (right panel). (C) Inhibitory effect of dominant-negative IRE1 on ER stress-induced mtHTT aggregation. After co-transfection with pHTTex120Q-GFP and either pcDNA or each dominant-negative mutant of ER sensor proteins (IRE1 DN, ATF6 DN and PERK DN), SH-SY5Y cells were left untreated or exposed to 1 mm thapsigargin (Tg). The aggregation of mtHTT was examined under ﬂuorescence microscope (left panel) and cell extracts were analyzed with western blotting using anti-HA, anti-GRP78 and anti-b-actin antibodies (right panel). Arrowheads indicate the expression of each construct.

Techniques Used: Activation Assay, Transfection, Microscopy, Western Blot, Control, Incubation, Dominant Negative Mutation, Cotransfection, Expressing, Construct

$Figure 1. Ectopic expression of IRE1 accumulates mtHTT aggregation. (A) Increased aggregation of mtHTT by IRE1 over-expression. AF5 cells were co-transfected with pHTTex120Q-GFP (mtHTT) and either pcDNA (PCD), pArfaptin2 (Arfaptin2) or pIRE1 (IRE1) for the indicated times and examined for the aggregation under ﬂuorescence microscope. Percentages of aggregation were determined by counting cells showing mtHTT aggregates among total GFP-positive cells. Bars represent mean values+SD from at least three inde- pendent experiments. P-values were calculated using t-test and were versus control (∗∗∗P , 0.001). (B) Detection of insoluble aggregates induced by IRE1. HEK293F cells were co-transfected with pHTTex120Q-GFP and either pcDNA, pArfaptin2 or pIRE1 for 36 h. Cell lysates were prepared, sepa- rated into soluble and insoluble fractions and analyzed by western blotting using anti-GFP or anti-a-tubulin antibody (upper panels). Cells were resus- pended in PBS and a ﬁlter trap assay was performed as described in Materials and Methods. The amounts of insoluble mtHTT-GFP aggregates on the blot were examined with western blot analysis using anti-GFP antibody (lower panel). (C) Stimulatory effect of IRE1 on mtHTT aggregation in primary neur- onal cells. Rat primary striatal and cortical neurons were cultured from embry- onic day 16 and maintained for 3 days in vitro. The striatal and cortical neurons were then co-transfected with pHTTex120Q-GFP and either pcDNA, pArfaptin2 or pIRE1 for 24 h. Bars represent mean values+SD (n ¼ 3). (D) Detergent-resistant aggregates of mtHTT formed by IRE1. SH-SY5Y cells were co-transfected with pHTTex120Q-GFP (green) and either pcDNA or pIRE1 for 24 h. Detergent-resistant aggregation assay was then performed using Triton X-100 and SDS (T/S) as described in Materials and Methods. Arrows indicate detergent-resistant aggregates of mtHTT.$
Figure Legend Snippet: Figure 1. Ectopic expression of IRE1 accumulates mtHTT aggregation. (A) Increased aggregation of mtHTT by IRE1 over-expression. AF5 cells were co-transfected with pHTTex120Q-GFP (mtHTT) and either pcDNA (PCD), pArfaptin2 (Arfaptin2) or pIRE1 (IRE1) for the indicated times and examined for the aggregation under ﬂuorescence microscope. Percentages of aggregation were determined by counting cells showing mtHTT aggregates among total GFP-positive cells. Bars represent mean values+SD from at least three inde- pendent experiments. P-values were calculated using t-test and were versus control (∗∗∗P , 0.001). (B) Detection of insoluble aggregates induced by IRE1. HEK293F cells were co-transfected with pHTTex120Q-GFP and either pcDNA, pArfaptin2 or pIRE1 for 36 h. Cell lysates were prepared, sepa- rated into soluble and insoluble fractions and analyzed by western blotting using anti-GFP or anti-a-tubulin antibody (upper panels). Cells were resus- pended in PBS and a ﬁlter trap assay was performed as described in Materials and Methods. The amounts of insoluble mtHTT-GFP aggregates on the blot were examined with western blot analysis using anti-GFP antibody (lower panel). (C) Stimulatory effect of IRE1 on mtHTT aggregation in primary neur- onal cells. Rat primary striatal and cortical neurons were cultured from embry- onic day 16 and maintained for 3 days in vitro. The striatal and cortical neurons were then co-transfected with pHTTex120Q-GFP and either pcDNA, pArfaptin2 or pIRE1 for 24 h. Bars represent mean values+SD (n ¼ 3). (D) Detergent-resistant aggregates of mtHTT formed by IRE1. SH-SY5Y cells were co-transfected with pHTTex120Q-GFP (green) and either pcDNA or pIRE1 for 24 h. Detergent-resistant aggregation assay was then performed using Triton X-100 and SDS (T/S) as described in Materials and Methods. Arrows indicate detergent-resistant aggregates of mtHTT.

Techniques Used: Expressing, Over Expression, Transfection, Microscopy, Control, Western Blot, TRAP Assay, Cell Culture, In Vitro

$Figure 3. Kinase activity of IRE1 is required for mtHTT aggregation. (A) Schematic diagram of functional domains and various mutants of IRE1. Asterisks indicate amino acid substitution of Lys599 with Ala in serine/threonine kinase domain and of Gly923 with Ala in endoribonuclease domain (left panel). HEK293F cells were transfected with pcDNA (PCD), pIRE1-HA or its mutant for 36 h and cell lysates were analyzed by western blotting using anti-p-IRE1, anti-HA and anti-a-tubulin antibodies. Arrowheads indicate the expression of IRE1 and its mutant and asterisk shows non-speciﬁc band, respectively. (B and C) Effects of IRE1 kinase activity on mtHTT aggregation. AF5 cells were co-transfected with pHTTex120Q-GFP and either pcDNA, pIRE1-HA or its mutant for the indicated times and then examined for the aggregation under ﬂuorescence microscope. Bars represent mean values+SD from at least three independent experiments. P-values were calculated using t-test and were versus control (∗P , 0.05; ∗∗∗P , 0.001) (B). HEK293F cells were co-transfected with pHTTex120Q-GFP and either pcDNA, pIRE1-HA or its mutant for 36 h. Cell lysates were separated into soluble and insoluble fractions and the fractions were subjected to western blotting using anti-GFP and anti-a-tubulin antibodies (C).$
Figure Legend Snippet: Figure 3. Kinase activity of IRE1 is required for mtHTT aggregation. (A) Schematic diagram of functional domains and various mutants of IRE1. Asterisks indicate amino acid substitution of Lys599 with Ala in serine/threonine kinase domain and of Gly923 with Ala in endoribonuclease domain (left panel). HEK293F cells were transfected with pcDNA (PCD), pIRE1-HA or its mutant for 36 h and cell lysates were analyzed by western blotting using anti-p-IRE1, anti-HA and anti-a-tubulin antibodies. Arrowheads indicate the expression of IRE1 and its mutant and asterisk shows non-speciﬁc band, respectively. (B and C) Effects of IRE1 kinase activity on mtHTT aggregation. AF5 cells were co-transfected with pHTTex120Q-GFP and either pcDNA, pIRE1-HA or its mutant for the indicated times and then examined for the aggregation under ﬂuorescence microscope. Bars represent mean values+SD from at least three independent experiments. P-values were calculated using t-test and were versus control (∗P , 0.05; ∗∗∗P , 0.001) (B). HEK293F cells were co-transfected with pHTTex120Q-GFP and either pcDNA, pIRE1-HA or its mutant for 36 h. Cell lysates were separated into soluble and insoluble fractions and the fractions were subjected to western blotting using anti-GFP and anti-a-tubulin antibodies (C).

Techniques Used: Activity Assay, Functional Assay, Transfection, Mutagenesis, Western Blot, Expressing, Microscopy, Control

Figure 5. IRE1 links ER stress-induced inhibition of autophagy ﬂux. (A) ER stress impairs autophagy ﬂux. SH-SY5Y cells were treated with DMSO or 1 mm thapsigargin (Tg) for 18 h and then exposed to 20 nM baﬁlomycin A1 (Baf.A1) for 6 h. Cells were then harvested and cell extracts were subjected to western blotting using the indicated antibodies. (B) Down-regulated IRE1 reduces ER stress-induced p62 accumulation. SH-SY5Y/pSuper-Neo (SH/Neo) and SH-SY5Y/ pSuper-shIRE1 (SH/shIRE1) stable cells were incubated with DMSO or 1 mm thapsigargin (Tg) for the indicated times and cell extracts were then analyzed with western blotting using anti-p62, anti-LC3, anti-p-IRE1, anti-GRP78 and anti-b-actin antibodies. (C) Down-regulation of IRE1 rescues thapsigargin-induced autophagy inhibition. SH-SY5Y/pSuper-Neo (SH/Neo) and SH-SY5Y/pSuper-shIRE1 (SH/shIRE1) cells were transfected with pmCherry-GFP-LC3 and incu- bated with DMSO or 1 mm thapsigargin (Tg) for 24 h. LC3 dots with GFP or mCherry signals were examined under confocal microscope (left panel). The cell numbers showing co-localization of both mCherry and GFP signals were counted and summarized with mean values+SD from at least three independent experiments (right panel). (D) Kinase activity of IRE1 contributes to autophagy inhibition. SH-SY5Y cells were transfected with either pcDNA, pIRE1-HA, pIRE1 DC-HA or pIRE1 DR-HA for 48 h and then harvested for western blot analysis using anti-p62, anti-LC3, anti-HA, anti-GRP78 and anti-b-actin anti- bodies. Cells exposed to 1 mm thapsigargin (Tg) for 24 h were used as a positive control. (E). Dominant-negative TRAF2 suppresses thapsigargin-induced autop- hagy inhibition. SH-SY5Y cells were transfected with pcDNA, pIRE1 DC-HA or pTRAF2 DN for 48 h and then incubated with DMSO or 1 mm thapsigargin (Tg) for 24 h. Cells were collected and analyzed with western blotting using the indicated antibodies.

Figure Legend Snippet: Figure 5. IRE1 links ER stress-induced inhibition of autophagy ﬂux. (A) ER stress impairs autophagy ﬂux. SH-SY5Y cells were treated with DMSO or 1 mm thapsigargin (Tg) for 18 h and then exposed to 20 nM baﬁlomycin A1 (Baf.A1) for 6 h. Cells were then harvested and cell extracts were subjected to western blotting using the indicated antibodies. (B) Down-regulated IRE1 reduces ER stress-induced p62 accumulation. SH-SY5Y/pSuper-Neo (SH/Neo) and SH-SY5Y/ pSuper-shIRE1 (SH/shIRE1) stable cells were incubated with DMSO or 1 mm thapsigargin (Tg) for the indicated times and cell extracts were then analyzed with western blotting using anti-p62, anti-LC3, anti-p-IRE1, anti-GRP78 and anti-b-actin antibodies. (C) Down-regulation of IRE1 rescues thapsigargin-induced autophagy inhibition. SH-SY5Y/pSuper-Neo (SH/Neo) and SH-SY5Y/pSuper-shIRE1 (SH/shIRE1) cells were transfected with pmCherry-GFP-LC3 and incu- bated with DMSO or 1 mm thapsigargin (Tg) for 24 h. LC3 dots with GFP or mCherry signals were examined under confocal microscope (left panel). The cell numbers showing co-localization of both mCherry and GFP signals were counted and summarized with mean values+SD from at least three independent experiments (right panel). (D) Kinase activity of IRE1 contributes to autophagy inhibition. SH-SY5Y cells were transfected with either pcDNA, pIRE1-HA, pIRE1 DC-HA or pIRE1 DR-HA for 48 h and then harvested for western blot analysis using anti-p62, anti-LC3, anti-HA, anti-GRP78 and anti-b-actin anti- bodies. Cells exposed to 1 mm thapsigargin (Tg) for 24 h were used as a positive control. (E). Dominant-negative TRAF2 suppresses thapsigargin-induced autop- hagy inhibition. SH-SY5Y cells were transfected with pcDNA, pIRE1 DC-HA or pTRAF2 DN for 48 h and then incubated with DMSO or 1 mm thapsigargin (Tg) for 24 h. Cells were collected and analyzed with western blotting using the indicated antibodies.

Techniques Used: Inhibition, Western Blot, Incubation, Transfection, Microscopy, Activity Assay, Positive Control, Dominant Negative Mutation

Figure 6. Inhibition of autophagy ﬂux by ER stress regulates mtHTT aggregation. (A and B) An increase in mtHTT aggregation by ER stress or autophagy inhibition was alleviated in IRE1 knock-down cells. SH-SY5Y/pSuper-Neo (SH/Neo) and SH-SY5Y/pSuper-shIRE1 (SH/shIRE1) cells were transfected with pHTTex120Q-GFP and exposed to DMSO or 1 mm thapsigargin (Tg) for 24 h in the presence or absence of 20 nM baﬁlomycin A1 (Baf.A1). Percentages of mtHTT aggregation were determined by counting cells showing aggregated pHTTex120Q-GFP under ﬂuorescence microscope (A). Cell extracts were prepared and analyzed with western blotting using anti-p62, anti-LC3, anti-p-IRE1, anti-GRP78 and anti-b-actin antibodies (B). Bars represent mean values+SD from at least three independent experiments. P-values were calculated using t-test and were versus control (∗P , 0.05; ∗∗∗P , 0.001). (C and D) ER stress-induced mtHTT aggregation is mediated via ATG5-dependent autophagy. M5-7 cells were incubated in the presence or absence of 10 ng/ml doxycycline (Dox.) for 4 days. After transfection with pHTTex120Q-GFP for 6 h, cells were exposed to DMSO or 30 nM thapsigargin (Tg) for additional 18 h. The aggregation of mtHTT was examined as in A (C). Cells extracts were subjected to western blotting (D). Bars represent mean values+SD (n ¼ 3).

Figure Legend Snippet: Figure 6. Inhibition of autophagy ﬂux by ER stress regulates mtHTT aggregation. (A and B) An increase in mtHTT aggregation by ER stress or autophagy inhibition was alleviated in IRE1 knock-down cells. SH-SY5Y/pSuper-Neo (SH/Neo) and SH-SY5Y/pSuper-shIRE1 (SH/shIRE1) cells were transfected with pHTTex120Q-GFP and exposed to DMSO or 1 mm thapsigargin (Tg) for 24 h in the presence or absence of 20 nM baﬁlomycin A1 (Baf.A1). Percentages of mtHTT aggregation were determined by counting cells showing aggregated pHTTex120Q-GFP under ﬂuorescence microscope (A). Cell extracts were prepared and analyzed with western blotting using anti-p62, anti-LC3, anti-p-IRE1, anti-GRP78 and anti-b-actin antibodies (B). Bars represent mean values+SD from at least three independent experiments. P-values were calculated using t-test and were versus control (∗P , 0.05; ∗∗∗P , 0.001). (C and D) ER stress-induced mtHTT aggregation is mediated via ATG5-dependent autophagy. M5-7 cells were incubated in the presence or absence of 10 ng/ml doxycycline (Dox.) for 4 days. After transfection with pHTTex120Q-GFP for 6 h, cells were exposed to DMSO or 30 nM thapsigargin (Tg) for additional 18 h. The aggregation of mtHTT was examined as in A (C). Cells extracts were subjected to western blotting (D). Bars represent mean values+SD (n ¼ 3).

Techniques Used: Inhibition, Knockdown, Transfection, Microscopy, Western Blot, Control, Incubation

Figure 7. IRE1 enhances mtHTT-mediated neuronal cell death via its kinase activity. (A) IRE1 knock-down reduces ER stress-induced mtHTT toxicity. SH-SY5Y/pSuper-Neo (SH/Neo) and SH-SY5Y/pSuper-shIRE1 (SH/shIRE1 #5 and #8) cells were transfected with pHTTex120Q-GFP and incubated with DMSO or 1 mm thapsigargin (Tg) for 24 h. Percentages of cell death were determined under ﬂuorescence microscope after staining with ethidium homodimer. (B) IRE1 increases the neurotoxicity of mtHTT. SH-SY5Y cells were co-transfected with pIRE1 and either pEGFP, pHTTex18Q-GFP (wtHTT) or pHTTex120Q-GFP (mtHTT) for 24 h. Percentages of cell death were determined under ﬂuorescence microscope after staining with ethidium homodimer as in (A). Bars represent mean values+SD (n ¼ 3). (C and D) Kinase activity of IRE1 contributes to death of STHdhQ111/111 cells. STHdhQ7/7 (Q7/7) and STHdhQ111/111 (Q111/111) cells were co-transfected with pEGFP and either pcDNA, pIRE1-HA, pIRE1 DC-HA or pIRE1 DR-HA for 72 h. Cells death was analyzed as in A (C) or cell extracts were prepared and subjected to western blotting using anti-caspase-12, anti-cleaved caspase-3, anti-p-IRE1, anti-HA and anti-b-actin antibodies (D). The arrowheads indicate speciﬁc signals detected by each antibody and the asterisks show non-speciﬁc signals. Bars represent mean values+SD (n ¼ 3). (E) STHdhQ111/111 striatal cells are more sensitive to ER stress-induced cell death than STHdhQ7/7 cells. STHdhQ7/7 (Q7/7) and STHdhQ111/111 (Q111/111) cells were co-transfected with pEGFP and either pcDNA or pIRE1 DC-HA for 48 h and then left untreated or exposed to DMSO or 100 nM thapsigargin (Tg) for additional 24 h. Cells death was analyzed as in (A). (F) Impaired autophagy activity increases mtHTT-mediated cell death under ER stress. M5-7 cells were cultured in the presence or absence of 10 ng/ml doxycycline (Dox.) for 4 days. After transfection with pEGFP or pHTTex120Q-GFP for 6 h, cells were exposed to DMSO or 30 nM thapsigargin (Tg) for additional 18 h. Cells death was analyzed as in (A). Bars represent mean values+SD from at least three independent experiments.

Figure Legend Snippet: Figure 7. IRE1 enhances mtHTT-mediated neuronal cell death via its kinase activity. (A) IRE1 knock-down reduces ER stress-induced mtHTT toxicity. SH-SY5Y/pSuper-Neo (SH/Neo) and SH-SY5Y/pSuper-shIRE1 (SH/shIRE1 #5 and #8) cells were transfected with pHTTex120Q-GFP and incubated with DMSO or 1 mm thapsigargin (Tg) for 24 h. Percentages of cell death were determined under ﬂuorescence microscope after staining with ethidium homodimer. (B) IRE1 increases the neurotoxicity of mtHTT. SH-SY5Y cells were co-transfected with pIRE1 and either pEGFP, pHTTex18Q-GFP (wtHTT) or pHTTex120Q-GFP (mtHTT) for 24 h. Percentages of cell death were determined under ﬂuorescence microscope after staining with ethidium homodimer as in (A). Bars represent mean values+SD (n ¼ 3). (C and D) Kinase activity of IRE1 contributes to death of STHdhQ111/111 cells. STHdhQ7/7 (Q7/7) and STHdhQ111/111 (Q111/111) cells were co-transfected with pEGFP and either pcDNA, pIRE1-HA, pIRE1 DC-HA or pIRE1 DR-HA for 72 h. Cells death was analyzed as in A (C) or cell extracts were prepared and subjected to western blotting using anti-caspase-12, anti-cleaved caspase-3, anti-p-IRE1, anti-HA and anti-b-actin antibodies (D). The arrowheads indicate speciﬁc signals detected by each antibody and the asterisks show non-speciﬁc signals. Bars represent mean values+SD (n ¼ 3). (E) STHdhQ111/111 striatal cells are more sensitive to ER stress-induced cell death than STHdhQ7/7 cells. STHdhQ7/7 (Q7/7) and STHdhQ111/111 (Q111/111) cells were co-transfected with pEGFP and either pcDNA or pIRE1 DC-HA for 48 h and then left untreated or exposed to DMSO or 100 nM thapsigargin (Tg) for additional 24 h. Cells death was analyzed as in (A). (F) Impaired autophagy activity increases mtHTT-mediated cell death under ER stress. M5-7 cells were cultured in the presence or absence of 10 ng/ml doxycycline (Dox.) for 4 days. After transfection with pEGFP or pHTTex120Q-GFP for 6 h, cells were exposed to DMSO or 30 nM thapsigargin (Tg) for additional 18 h. Cells death was analyzed as in (A). Bars represent mean values+SD from at least three independent experiments.

Techniques Used: Activity Assay, Knockdown, Transfection, Incubation, Microscopy, Staining, Western Blot, Cell Culture

Review

gfp (Santa Cruz Biotechnology)

Structured Review

Images

1) Product Images from "IRE1 plays an essential role in ER stress-mediated aggregation of mutant huntingtin via the inhibition of autophagy flux."

Similar Products

Image Search Results