Antibodies
The following primary antibodies were used: mouse anti-CCDC134 (Santa Cruz Biotechnology, #sc-393390, RRID: AB_3662100, 1:500); mouse anti-GRP94 (R&D Systems, #MAB7606, RRID: AB_3644153, 1:2,000); mouse anti-GRP94 (Santa Cruz Biotechnology, #sc-393402, RRID: AB_2892568, 1:2,000); rabbit anti-LRP6 (Cell Signaling Technology, #2560, RRID: AB_2139329, 1:1,000); mouse anti-α-tubulin (MilliporeSigma, #T6199, RRID: AB_477583, 1:10,000); mouse anti-α-tubulin (Abcam, #ab11304, RRID: AB_297909, 1:1,000); mouse anti-STT3A (Abnova, #H00003703-M02, RRID: AB_530104, 1:1,000); rabbit anti-STT3A (Proteintech, #12034-1-AP, RRID: AB_2877818, 1:1,000); rabbit anti-IGF1Rβ (Cell Signaling Technology, #9750, RRID: AB_10950969, 1:1,000); mouse anti-GAPDH (Proteintech, #60004-1-Ig, RRID: AB_2107436, 1:10,000); rabbit anti-PSAP (GeneTex, #GTX101064, RRID: AB_2037779, 1:1,000); mouse anti-HA (GenScript, #A01244, RRID: AB_1289306, 1:1,000); rabbit anti-HA (Bethyl, #A191-102, RRID: AB_2891412, 1:2,000); rabbit anti-uL22 (Abcepta, #AP9892b, RRID: AB_10613776, 1:1,000); rabbit anti-uL2 (Abcam, #ab169538, RRID: AB_2714187, 1:1,000); rabbit anti-SEC61β (Cell Signaling Technology, #14648, RRID: AB_2798555, 1:1,000); rabbit anti-TRAPα (MilliporeSigma, #HPA011276, RRID: AB_1857503, 1:1,000); rabbit anti-STT3B (Proteintech, #15323-1-AP, RRID: AB_2198046, 1:1,000); rabbit anti-FKBP11 (Atlas Antibodies, #HPA041709, RRID: AB_10794487, 1:1,000); mouse anti-FLAG (MilliporeSigma, #F1804, RRID: AB_262044, 1:1,000); mouse anti-BiP (BD Transduction Laboratories, #610978, RRID: AB_398291, 1:1000).
The following secondary antibodies conjugated to horseradish peroxidase were used: Peroxidase AffiniPure Donkey Anti-Mouse IgG (H+L) (Jackson ImmunoResearch Laboratories, #715-035-150, RRID: AB_2340770, 1:10,000); Peroxidase AffiniPure Donkey Anti-Rabbit IgG (H+L) (Jackson ImmunoResearch Laboratories, #111-035-144, RRID: AB_2307391, 1:10,000); Peroxidase Donkey Anti-Rabbit IgG (Fc specific) (Sigma-Aldrich, #SAB3700863, RRID: AB_3675584, 1:10,000); Peroxidase Rabbit Anti-Mouse IgG H&L (Abcam, #ab6728, RRID: AB_955440, 1:10,000). Secondary antibodies conjugated to IRDye 800CW were obtained from LI-COR (IRDye 800CW Donkey anti-Mouse IgG Secondary Antibody, 1:10,000).
Constructs
CCDC134 constructs
Doxycycline-inducible 3xFLAG–CCDC134 (tag inserted after the ER signal sequence) was cloned by polymerase chain reaction (PCR) amplification followed by Gibson assembly (New England Biolabs) into pcDNA5-FRT/TO. Doxycycline-inducible 3xHA–CCDC134 (tag inserted after the ER signal sequence) mutants were cloned by PCR amplification followed by Gibson assembly (New England Biolabs) into pLenti-TRE-rtta3G-BLAST4.
GRP94 constructs
pENTR2B-3xFLAG-GRP94 (wild type) and pENTR2B-3xFLAG-GRP945N (includes mutations in all five facultative sequons: S64A, S109A, S447A, T483I, T504I)4 were used as templates to clone all mutant constructs used to generate RKO addback cell lines. All mutant constructs were generated using PCR amplification followed by Gibson assembly and cloned into pLenti CMV Puro DEST63 using gateway methods. Doxycycline-inducible 3xFLAG–GRP94 (tag inserted after the ER signal sequence) constructs for transient expression in HEK293 cells were cloned by PCR amplification followed by Gibson assembly into pcDNA5/FRT/TO. These were used as templates to generate mutant constructs by QuikChange Site-Directed Mutagenesis (Agilent, 210519, 200514).
STT3A constructs
pENTR2B-STT3A-FLAG4 was used as a template to generate STT3A mutants using PCR amplification followed by Gibson assembly and cloned into pLenti CMV Puro DEST63 using Gateway methods. All constructs were fully sequenced to confirm accuracy.
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