Anti-IRE1 antibody [9F2]

• Flow Cyt

Unknown

Flow Cytometry - Anti-IRE1 antibody [9F2] (AB96481)

Overlay histogram showing Raji cells stained with ab96481 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab96481, 1/100 dilution) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2μg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in Raji cells fixed with 4% paraformaldehyde (10 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-IRE1 antibody [9F2] (AB96481)

Immunohistochemical analysis of paraffin-embedded Human brain tissue (A) and stomach tissue (B), showing cytoplasmic localization using ab96481 at 1/200 dilution with DAB staining.

• WB

Unknown

Western blot - Anti-IRE1 antibody [9F2] (AB96481)

All lanes:

Western blot - Anti-IRE1 antibody [9F2] (ab96481) at 1/500 dilution

Lane 1:

Raji cell lysate

Lane 2:

A431 cell lysate

Lane 3:

Jurkat cell lysate

Lane 4:

HeLa cell lysate

Lane 5:

HEK293 cell lysate

Predicted band size: 110 kDa

false

• WB

CiteAb

Western blot - Anti-IRE1 antibody [9F2] (AB96481)

Western Blotting using Anti-IRE1 antibody [9F2], ab96481. Publication image from Jeon, Y. J. et al., 2018, Nat Commun, 30504895. Legend direct from paper.

TUSC3 deficiency selectively modulates Unfolded Protein Responses. a GSEA plots showing upregulation of the genes involved in Unfolded Protein Reponses. b Western blot analyses showing weakened IRE1α-XBP1 and PERK-EIF2α pathways in A549 TUSC3 KO cells whereas enhanced in TUSC3/HRD1 DKO cells in response to ER stress induction. c Subcellular fractionation assay showing enhanced chromatin-bound ATF6α in A549 TUSC3 KO and HRD1/TUSC3 DKO cells. The indicated cells were stimulated by 1.0 uM of TG for 6 or 9 h, respectively. Anti-Calnexin or anti-PARP1 antibody was used for ER/Golgi or Nuclear fraction markers, respectively. d Increased ATF6α-dependent ER heat-shock proteins in TUSC3 deficient cells. The cells were transfected by scrambled or siATF6α siRNAs for 48 h followed by exposing to DMSO or TM (3 ug/ml, 16 h). Western blot analysis shows elevated expression of GRP78 and GRP94 in A549 TUSC3 KO cells. A mitochondrial heat-shock protein, GRP75 was used for negative control. e Schematic diagram showing primary structure of TUSC3 protein and its mutants (TUSC3 CCSS). CSVC indicates the amino acids in C-X-X-C motif. f Rescued IRE1α and PERK expression by reconstitution of TUSC3 or its CCSS mutant. g Restored nuclear localization of the ATF6α protein by the reconstitution of TUSC3 but not by CCSS mutant in A549 TUSC3 KO cells. h Rescued the colonization ability of H460 TUSC3KO cells by suppressing ATF6α expression. 1 x 106 of the control cells or ATF6α knock-downed TUSC3 KO ells was intravenously injected into four NOD scid gamma mice. p-value was calculated by unpaired student t-test (*p = 0.002). The data for the tumor area from the control cells-injected mice are shared with Fig. 5g. i Co-expression analysis of ATF6α with TUSC3 protein showing inverse correlation between TUSC3 and ATF6α activation in lung cancer patient samples. p-value was obtained by Chi square analysis. The scale bar is shown as 150 µm. j Rescued colonization ability of TUSC3/HRD1 DKO cells. The tumor area was calculated as the total area of lung occupied by cancer is field of view using Image J software. The region for the cancer was expressed as percentage. p-value was calculated by unpaired student t-test (*p < 0.001)

false

• WB

CiteAb

Western blot - Anti-IRE1 antibody [9F2] (AB96481)

Western Blotting using Anti-IRE1 antibody [9F2], ab96481. Publication image from Jeon, Y. J. et al., 2018, Nat Commun, 30504895. Legend direct from paper.

TUSC3 deficiency selectively modulates Unfolded Protein Responses. a GSEA plots showing upregulation of the genes involved in Unfolded Protein Reponses. b Western blot analyses showing weakened IRE1α-XBP1 and PERK-EIF2α pathways in A549 TUSC3 KO cells whereas enhanced in TUSC3/HRD1 DKO cells in response to ER stress induction. c Subcellular fractionation assay showing enhanced chromatin-bound ATF6α in A549 TUSC3 KO and HRD1/TUSC3 DKO cells. The indicated cells were stimulated by 1.0 uM of TG for 6 or 9 h, respectively. Anti-Calnexin or anti-PARP1 antibody was used for ER/Golgi or Nuclear fraction markers, respectively. d Increased ATF6α-dependent ER heat-shock proteins in TUSC3 deficient cells. The cells were transfected by scrambled or siATF6α siRNAs for 48 h followed by exposing to DMSO or TM (3 ug/ml, 16 h). Western blot analysis shows elevated expression of GRP78 and GRP94 in A549 TUSC3 KO cells. A mitochondrial heat-shock protein, GRP75 was used for negative control. e Schematic diagram showing primary structure of TUSC3 protein and its mutants (TUSC3 CCSS). CSVC indicates the amino acids in C-X-X-C motif. f Rescued IRE1α and PERK expression by reconstitution of TUSC3 or its CCSS mutant. g Restored nuclear localization of the ATF6α protein by the reconstitution of TUSC3 but not by CCSS mutant in A549 TUSC3 KO cells. h Rescued the colonization ability of H460 TUSC3KO cells by suppressing ATF6α expression. 1 x 106 of the control cells or ATF6α knock-downed TUSC3 KO ells was intravenously injected into four NOD scid gamma mice. p-value was calculated by unpaired student t-test (*p = 0.002). The data for the tumor area from the control cells-injected mice are shared with Fig. 5g. i Co-expression analysis of ATF6α with TUSC3 protein showing inverse correlation between TUSC3 and ATF6α activation in lung cancer patient samples. p-value was obtained by Chi square analysis. The scale bar is shown as 150 µm. j Rescued colonization ability of TUSC3/HRD1 DKO cells. The tumor area was calculated as the total area of lung occupied by cancer is field of view using Image J software. The region for the cancer was expressed as percentage. p-value was calculated by unpaired student t-test (*p < 0.001)

false