KIAA1324 Mutant Expression and Analysis

KIAA1324 and its mutants were cloned into the pCMV-3HA vector (Clontech, California, USA) as previously described [13 (link)]. Asp (N) to Gln (Q) amino acid exchange mutation in putative N-glycosylation sites was performed by a DpnI-site directed mutagenesis method using mutagenic primers [21 (link)]. Anti-HA (Santa Cruz Biotechnology, Texas, USA, sc-7392) and anti-PARP (Santa Cruz Biotechnology, sc-8007), anti-caspase-3 (Cell Signaling Technology, Massachusetts, USA, #9662), anti-caspase-7 (Cell Signaling Technology, #9494), anti-phosphor-JNK (Cell Signaling Technology, #4668), anti-JNK (Cell Signaling Technology, #9252), anti-GRP78 (Abcam, Cambridge, United Kingdom, ab21685), anti-PDI, anti-β-actin (Abcam, ab8227), anti-KIAA1324 (Sigma, Missouri, USA, SAB2900912) and α-tubulin (Sigma, T5168) antibodies were used. 2F-Peracetyl-Fucose (Sigma, #344827), SP600125 (Sigma, S5567), SB203580 (Sigma, S8307), U0126 (Sigma, U120), tunicamycin (Sigma, T7765), and doxycycline (Sigma, #24390-14-5) chemicals were used.

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Yun R., Hong E., Kim J., Park B., Kim S.J., Lee B., Song Y.S., Kim S.J., Park S, & Kang J.M. (2023). N-linked glycosylation is essential for anti-tumor activities of KIAA1324 in gastric cancer. Cell Death & Disease, 14(8), 546.

Publication 2023

Anti-HA anti-PARP anti-caspase-3 anti-caspase-7 anti-phosphor-JNK anti-JNK anti-GRP78 anti-β-actin α-tubulin 2F-Peracetyl-Fucose SP600125 SB203580 U0126 tunicamycin doxycycline

Actin Amino acid Antibodies Caspase 3 Caspase 7 Doxycycline Fucose Glycosylation Grp78 Mutagenic Mutation Phosphor Primers Sb203580 Site directed mutagenesis Sp600125 Tunicamycin U0126 Vector α tubulin

Corresponding Organization : University Hospitals of Cleveland

Other organizations : Soongsil University, WellSpan York Hospital, Tsukuba International University, University of Tsukuba, Case Western Reserve University, University School, Seoul National University, Boditech Med (South Korea)

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Variable analysis

independent variables

Asp (N) to Gln (Q) amino acid exchange mutation in putative N-glycosylation sites

dependent variables

Not explicitly mentioned

control variables

KIAA1324 and its mutants were cloned into the pCMV-3HA vector
Anti-HA, anti-PARP, anti-caspase-3, anti-caspase-7, anti-phosphor-JNK, anti-JNK, anti-GRP78, anti-PDI, anti-β-actin, anti-KIAA1324, and α-tubulin antibodies were used
2F-Peracetyl-Fucose, SP600125, SB203580, U0126, tunicamycin, and doxycycline chemicals were used

controls

Positive control: Not specified
Negative control: Not specified

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