GRP78/HSPA5 Antibody

Detection of GRP78/HSPA5 by Western Blot

Increased autophagy and ER stress in A673 cells. WE-68 and A673 cells were treated with 15–45 nM of AUY922, 2 µM of VE821, 5 µM of KU55933 and their combinations. DMSO was used for control. a, b, d, e Analysis of indicated proteins was done by Western blot after 24 h; vinculin and beta -actin were used to control protein loading. Immunoblots are representative of at least two independent experiments. c, f Quantifications of selected proteins Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/33743824), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of GRP78/HSPA5 by Western Blot

IL-22BPi1 and IL-22BPi2, but not IL-22BPi3, are bona fide client proteins of GRP78. (A,B) Identification of GRP78, GRP94, GRP170 and calnexin in interactomes of IL-22BPi1 and IL-22BPi2. Silver-stained (S.S) gels of proteins (co-)immunoprecipitated (IP) with FLAG resins from HEK293 or HeLa cells transiently transfected with the indicated expression plasmids or control empty vector (EV) are shown. Numbered arrow-heads indicate proteins identified by mass spectrometry included in the inset summary Table (detailed information in Supplementary Data 2). Co-immunoprecipitated proteins without DSP treatment were immunoblotted for detection by FLAG Ab. Asterisk, IL-22BPi1; empty arrowhead, IL-22; solid rhombus, IL-22BPi2; empty rhombus, IL-2EX. (Inset) Summary table with the mass spectrometry results from protein identification. # Unique peptides, # Peptides, and # PSMs are: the number of peptide sequences unique to a protein group, the number of distinct peptide sequences in the protein group and the total number of identified peptide sequences (peptide spectrum matches) for the protein. (C) IL-22BPi1, IL-22BPi2 and IL-2EX4 bind the substrate-binding domain of GRP78. HEK293 cells were transiently transfected with the indicated constructs and 24 h later were either treated with 100 ng/ml Mut SubAB or SubAB for 3 h. Cell lysates (CL) were co-immunoprecipitated with FLAG agarose. CL and IP proteins were immunoblotted for FLAG or GRP78. (D) HEK293 cells were individually transfected with expression plasmids for the three IL-22BP isoforms together with either GRP78 or GRP78T37G expression vectors or empty vector (EV). 24 h later secreted protein was measured by ELISA for IL-22BP (mean ± SEM; n = 3; *p < 0.05, **p < 0.01 by unpaired t-test). Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/30619294), licensed under a CC-BY license. Not internally tested by R&D Systems.