Fig 1: Remote ischemic preconditioning (RIPC) protects ErbB2 (Erb-B2 receptor tyrosine kinase 2) from ischemia/reperfusion (I/R)-mediated degradation and the loss of ErbB2 results in Trx2 (thioredoxin 2) autophagy and apoptosis. A, Mice were subjected to a sham procedure, 30 min ischemia, I/R, RIPC or RIPC+I/R, and the infarcted tissue region were excised. Protein extract was prepared from the excised tissue and analyzed by Western blotting for ErbB2, Trx2, catalase, SOD1, SOD2, and total actin. B, ErbB2 and Trx2 levels in the blots were quantified and plotted as a bar graph (n=3). *P<0.01 vs sham; **P<0.01 vs I/R. C, HCAECs were transfected with nontarget (NT) or ErbB2 siRNA (25, 50, 100 nmol/L). After 48 h of transfection, cell lysates were prepared, and an equal amount of protein from each cell lysate was analyzed for Trx1, Trx2, SOD1, SOD2, and catalase levels using their specific antibodies. Blots were reprobed with anti-ErbB2 and anti-ß-actin antibodies to determine the depletion of ErbB2 and equal protein loading, respectively. Bar graph shows the level of Trx2. D, HCAECs were transfected with pcDNA3 empty or pcDNA3-ErbB2 plasmids. After 24 h of transfection, cells were exposed to hypoxia/reoxygenation (H/R), and cell lysates were prepared. An equal amount of protein from each sample was analyzed for Trx2 and ErbB2 levels using their specific antibodies. Blots were reprobed with anti-ß-actin antibodies. E, HCAECs were exposed to normoxia or H/R, mRNA was isolated, and cDNA was synthesized. PCR was performed to determine levels of ErbB2 (primers, 5'- GTGCTGGTCAAGAGTCCCAACCATG-3' and 5'- ATCTGGCTGGTTCACATATTCAGGC-3’), Trx2 (primers, 5'- CCACACCTTGGTCCTCATCT-3' and 5'- AGGAGGTGGAAGGGATGACT-3'), and ß-actin (primers, 5'-CCGCCAGCTCACCAT-3' and 5'-GTGTGGTGCCAGATTTTCTC-3'). F, HCAECs were treated with 3-methyl adenine (3-MA), MG132 (proteasomal inhibitor), or chloroquine (CLQ) before exposing them to H/R. After the treatment, cells were lysed, and the cell lysates were analyzed by Western blotting for Trx2, p62, microtubule-associated protein 1, light chain 3-I/II level using its specific antibody, and the blot was reprobed for ß-actin for normalization. G, HCAECs were transfected with Ambra1 or beclin 1 (BCLN1) siRNA in combination with NT or ErbB2 siRNA (100 nmol/L). After 48 h of transfection, cell lysates were prepared, and an equal amount of protein from each cell lysate was analyzed for Trx2, beclin 1, and GAPDH levels using their specific antibodies. H, HCAECs were transfected with NT or ATG5 (autophagy-related 5) siRNA and exposed to H/R or transfected with ATG5 siRNA and ErbB2 siRNA. After 48 h of transfection and H/R treatment, cell lysates were prepared, and an equal amount of protein from each cell lysate was analyzed for Trx2, ATG5, ErbB2, and GAPDH levels using their specific antibodies. I, HCAECs were exposed to normoxia or H/R, and cell lysates were immunoprecipitated with anti-ErbB2 antibodies. Immunoprecipitates were analyzed for ATG5, AMBRA1 (activating molecule in beclin 1-regulated autophagy), and ErbB2. J, To study the in situ interaction of ErbB2 and ATG5, HCAECs were subjected to H/R and proximity ligation assay (PLA) was performed using anti-ErbB2 and anti-ATG5 antibodies. Scale bar=20 mm. K, Green foci-proximity signals of ErbB2 and ATG5 association were counted and plotted as a bar graph. *P<0.01 vs normoxia. Student t test. L, HCAECs were exposed to normoxia or H/R, and cell lysates were immunoprecipitated with anti-pTyr antibodies. Immunoprecipitates were analyzed for ATG5 and AMBRA1. M, HCAECs were transfected with NT or Trx2 siRNA (100 nmol/L), and after recovery from transfection, they were treated with or without preconditioned medium (PM) and exposed to H/R. Cells undergoing apoptosis were labeled with annexin V-FITC conjugate, and the percentage of apoptotic cells was quantitated and plotted as a bar graph (n=3). *P<0.01 vs NT siRNA normoxia, **P<0.01 vs NT siRNA H/R, †P<0.01 vs NT siRNA PM+H/R. One way ANOVA was performed using GraphPad-Prism software (version 8).