NOX2-Dependent Reactive Oxygen Species Regulate Formyl-Peptide Receptor 1-Mediated TrkA Transactivation in SH-SY5Y Cells

<div>FPR1-mediated TrkA transactivation triggers PLC<i>γ</i>1/PKC pathway activation. SH-SY5Y cells were serum-deprived for 24 hours and (a) stimulated for 2, 5, or 10 minutes with N-fMLP or pretreated with PTX or cyclosporin H before the stimulation for 5 minutes with N-fMLP. (b) Cells were also preincubated with apocynin or rottlerin before N-fMLP stimulation. Fifty micrograms of membrane lysates was immunoblotted with (a) an anti-PKC<i>δ</i> (<i>α</i>-PKC<i>δ</i>) or anti-PKC<i>α</i> (<i>α</i>-PKC<i>α</i>) antibody or (b) with an anti-phosphoPKC<i>δ</i> (T507) (<i>α</i>-pPKC<i>δ</i>(T507)) antibody. An anti-CD133 (<i>α</i>-CD133) or an anti-tubulin (<i>α</i>-tubulin) antibody was used as a control for protein loading. <svg height="13.532pt" id="M17" style="vertical-align:-3.4295pt" version="1.1" viewbox="-0.0498162 -10.1025 50.6052 13.532" width="50.6052pt" xmlns="http://www.w3.org/2000/svg" xmlns:xlink="http://www.w3.org/1999/xlink"><g transform="matrix(.0091,0,0,-0.0091,0,-5.741)"><path d="M486 158C486 177 478 202 466 220C413 228 386 236 336 262C386 288 413 297 466 304C478 323 486 347 485 366C470 376 444 381 422 380C389 338 368 319 321 288C323 345 329 372 349 422C339 442 322 461 305 470C289 461 271 442 262 422C281 372 287 345 290 288C243 319 222 338 189 380C167 381 142 376 125 366C125 347 133 322 145 304C198 296 225 288 275 262C225 236 198 227 145 220C133 201 125 177 126 158C141 148 167 143 189 144C222 186 243 205 290 236C288 179 282 152 262 102C272 82 289 63 306 54C322 63 340 82 350 102C330 152 324 179 321 236C368 205 390 186 422 144C444 143 470 148 486 158Z"></path></g><g transform="matrix(.013,0,0,-0.013,6.079,0)"><path d="M570 304C570 398 525 448 414 448C385 448 343 445 312 434L329 511L321 518C297 504 262 482 244 460L233 411C195 397 159 381 128 358L135 332C160 347 189 360 224 373L111 -147C97 -210 84 -218 17 -231L13 -257L254 -247L259 -218L233 -216C183 -212 177 -202 189 -142L218 -1C238 -10 266 -12 283 -12C351 3 429 48 483 105C543 168 570 242 570 304ZM482 289C482 161 380 33 304 33C278 33 248 51 233 69L303 396C326 400 352 403 369 403C428 403 482 380 482 289Z"></path></g><g transform="matrix(.013,0,0,-0.013,17.421,0)"><path d="M512 -3V55L134 254V256L512 456V514L75 281V230L512 -3Z"></path></g><g transform="matrix(.013,0,0,-0.013,28.684,0)"><path d="M241 635C89 635 35 457 35 312C35 153 89 -12 240 -12C390 -12 443 166 443 312C443 466 390 635 241 635ZM238 602C329 602 354 454 354 312C354 172 330 22 240 22C152 22 124 173 124 313S148 602 238 602Z"></path></g><g transform="matrix(.013,0,0,-0.013,34.924,0)"><path d="M113 -12C146 -12 170 11 170 46C170 78 146 103 114 103S58 78 58 46C58 11 82 -12 113 -12Z"></path></g><g transform="matrix(.013,0,0,-0.013,37.888,0)"><path d="M241 635C89 635 35 457 35 312C35 153 89 -12 240 -12C390 -12 443 166 443 312C443 466 390 635 241 635ZM238 602C329 602 354 454 354 312C354 172 330 22 240 22C152 22 124 173 124 313S148 602 238 602Z"></path></g><g transform="matrix(.013,0,0,-0.013,44.128,0)"><path d="M153 550H386L412 615L406 623H120L82 318C104 327 142 338 184 338C294 338 347 275 347 187C347 112 305 39 221 39C160 39 119 71 97 89C88 97 80 96 71 90C59 80 50 67 49 57C48 45 52 36 66 23C80 9 123 -12 169 -12C221 -11 288 15 342 59C403 109 431 165 431 225C431 308 366 395 238 395C212 395 165 379 127 364L153 550Z"></path></g></svg> compared to unstimulated cells. <sup>§</sup><svg height="11.7782pt" id="M18" style="vertical-align:-3.42938pt" version="1.1" viewbox="-0.0498162 -8.34882 44.5261 11.7782" width="44.5261pt" xmlns="http://www.w3.org/2000/svg" xmlns:xlink="http://www.w3.org/1999/xlink"><g transform="matrix(.013,0,0,-0.013,0,0)"><path d="M570 304C570 398 525 448 414 448C385 448 343 445 312 434L329 511L321 518C297 504 262 482 244 460L233 411C195 397 159 381 128 358L135 332C160 347 189 360 224 373L111 -147C97 -210 84 -218 17 -231L13 -257L254 -247L259 -218L233 -216C183 -212 177 -202 189 -142L218 -1C238 -10 266 -12 283 -12C351 3 429 48 483 105C543 168 570 242 570 304ZM482 289C482 161 380 33 304 33C278 33 248 51 233 69L303 396C326 400 352 403 369 403C428 403 482 380 482 289Z"></path></g><g transform="matrix(.013,0,0,-0.013,11.342,0)"><path d="M512 -3V55L134 254V256L512 456V514L75 281V230L512 -3Z"></path></g><g transform="matrix(.013,0,0,-0.013,22.605,0)"><path d="M241 635C89 635 35 457 35 312C35 153 89 -12 240 -12C390 -12 443 166 443 312C443 466 390 635 241 635ZM238 602C329 602 354 454 354 312C354 172 330 22 240 22C152 22 124 173 124 313S148 602 238 602Z"></path></g><g transform="matrix(.013,0,0,-0.013,28.845,0)"><path d="M113 -12C146 -12 170 11 170 46C170 78 146 103 114 103S58 78 58 46C58 11 82 -12 113 -12Z"></path></g><g transform="matrix(.013,0,0,-0.013,31.809,0)"><path d="M241 635C89 635 35 457 35 312C35 153 89 -12 240 -12C390 -12 443 166 443 312C443 466 390 635 241 635ZM238 602C329 602 354 454 354 312C354 172 330 22 240 22C152 22 124 173 124 313S148 602 238 602Z"></path></g><g transform="matrix(.013,0,0,-0.013,38.049,0)"><path d="M153 550H386L412 615L406 623H120L82 318C104 327 142 338 184 338C294 338 347 275 347 187C347 112 305 39 221 39C160 39 119 71 97 89C88 97 80 96 71 90C59 80 50 67 49 57C48 45 52 36 66 23C80 9 123 -12 169 -12C221 -11 288 15 342 59C403 109 431 165 431 225C431 308 366 395 238 395C212 395 165 379 127 364L153 550Z"></path></g></svg> compared to N-fMLP-stimulated cells.</div>

Oxidative Medicine and Cellular Longevity

fig5

Figure 5

Figure 5: NOX2-Dependent Reactive Oxygen Species Regulate Formyl-Peptide Receptor 1-Mediated TrkA Transactivation in SH-SY5Y Cells 