Fmlp Gradient 1 Μ M 10 0 Nm 10 Nm 1 Nm IL-8 Gradient Xu Et Al

A B Total path length (mm) 100 150 200 250 300 50 0 1 mM 100 nM 10 nM 1 nM NG NG fMLP 1 nM 10 nM 100 nM 1 uM NG fMLPgradient IL 1 nM IL8 - 8 10 nM 100 nM 11 muMM Speed (mm/min) 10 15 20 25 30 0 5 NG fMLP 1 nM 10 nM 100 nM 1 uM NG 1 nM IL8 IL 10 nM - 8 100 nM 1 muMM Directionality 0.2 0.4 0.6 0.8 1.2 IL 0 1 - 8 gradient NG fMLP 1 nM 10 nM 100 nM 1 uM NG S1 Fig. etal., Xu IL 1 nM IL8 - 8 10 nM 100 nM 1 muMM Xu et al., Figure S2 Differentiation A Mm Hs B 150kD P120 GAP 102kD 0 1 2 3 4 5 (days) 150kD 80kD CAPRI 100kD p120 GAP CAPRI 80kD RASAL1 76kD 150kD 76kD RASAL1 RASAL2 RASAL2 102kD 150kD 150kD RASAL3 150kD 102kD CD11 150kD 102kD CD11 50kD actin actin 38kD 38kD B A Merge CAAX-mCh Mem-cer Merge CAAX-mCh Mem-cer Merge CAAX-mCh C1AC1A-YFP Merge CAAX-mCh C1AC1A-YFP 80s 90s 100s 110s 120s 130s 140s 140s 130s 120s 110s 100s 90s 80s 0s 80s 90s 100s 110s 120s 130s 140s 140s 130s 120s 110s 100s 90s 80s 0s 10s 20s 30s 40s 50s 60s 60s 50s 40s 30s 20s 10s 0s 0s 10s 20s 30s 40s 50s 60s 60s 50s 40s 30s 20s 10s 0s 80s 90s 100s 110s 120s 130s 140s 140s 130s 120s 110s 100s 90s 80s 0s 10s 20s 30s 40s 50s 60s 60s 50s 40s 30s 20s 10s 0s Xu et al., Figure S3 etal., Xu 16 70s 70s 16 0s 0s 16 70s 0s Xu et al., Figure S4 PM 0s 2.5s 5s 7.5s 10s 15s 20s CAPRI caprikd/OE RBD Merge PM 25s 30s 35s 40s 50s 60s 70s CAPRI RBD Merge Merge PM CAPRI Merge PM CAPRI 30s 35s 40s 45s 50s 60s 60s 50s 45s 40s 35s 30s 0s 5s 10s 15s 20s 25s 25s 20s 15s 10s 5s 0s Xu et al., Figure S5 etal., Xu Xu et al., Figure S6 No treatment No treatment 100 nM PT GFP PM Merge CAPRI PM Merge CAPRI PM Merge 0s 0s 0s 10s 10s 10s 10s10s 20s 20s 20s 20s20s 30s 30s 30s 60s120s 40s 40s 40s 60s 60s 60s Xu et al., Figure S7 Hs NF1 Hs CAPRI Mm CAPRI Hs NF1 Hs CAPRI Mm CAPRI Hs NF1 Hs CAPRI Mm CAPRI Hs NF1 Hs CAPRI Mm CAPRI Hs NF1 Hs CAPRI Mm CAPRI Hs NF1 Hs CAPRI Mm CAPRI Hs NF1 Hs CAPRI Mm CAPRI Hs NF1 Hs CAPRI Mm CAPRI Xu et al., Figure S8 0s 4s 6s 8s 10s 12s 14s 16s 18s 20s 22s 24s 26s 28s 30s 32s 34s 36s 38s 40s 42s 44s 46s 48s 50s 52s 54s 56s 58s 60s 62s 64s 66s 68s 70s 72s 74s 76s 78s 80s 82s 84s 86s 88s WT - 90s 92s 94s 96s 98s 100s 102s 104s 106s 108s 110s PM CAPRI 112s 114s 116s 118s 120s 122s 124s 126s 128s 130s 132s 134s 136s 138s 140s 142s 144s 146s 148s 150s 160s 162s 164s 166s 168s 170s 172s 174s 176s 178s 180s 182s 184s 0s 4s 6s 8s 10s 12s 14s 16s 18s 20s 22s 24s 26s 28s 30s 32s 34s 36s 38s 40s 42s 44s 46s 48s 50s 52s 54s 56s 58s 60s 62s 64s 66s 68s 70s 72s 74s 76s 78s 80s 82s 84s 86s 88s R472A - 90s 92s 94s 96s 98s 100s 102s 104s 106s 108s 110s PM 112s 114s 116s 118s 120s 122s 124s 126s 128s 130s 132s CAPRI 134s 136s 138s 140s 142s 144s 146s 148s 150s 160s 162s 164s 166s 168s 170s 172s 174s 176s 178s 180s 182s 184s 0s 4s 6s 8s 10s 12s 14s 16s 18s 20s 22s 24s 26s 28s 30s 32s 34s 36s 38s 40s 42s 44s 46s 48s 50s 52s 54s 56s 58s 60s 62s 64s 66s PH - 68s 70s 72s 74s 76s 78s 80s 82s 84s 86s 88s PM 90s 92s 94s 96s 98s 100s 102s 104s 106s 108s 110s CAPRI 112s 114s 116s 118s 120s 122s 124s 126s 128s 130s 132s 134s 136s 138s 140s 142s 144s 146s 148s 150s 160s 162s 164s 166s 168s 170s 172s 174s 176s 178s 180s 182s 184s 0s 4s 6s 8s 10s 12s 14s 16s 18s 20s 22s 24s 26s 28s 30s 32s 34s 36s 38s 40s 42s 44s 46s 48s 50s 52s 54s 56s 58s 60s 62s 64s 66s PH 68s 70s 72s 74s 76s 78s 80s 82s 84s 86s 88s - C2 - 90s 92s 94s 96s 98s 100s 102s 104s 106s 108s 110s PM CAPRI 112s 114s 116s 118s 120s 122s 124s 126s 128s 130s 132s CAPRI 134s 136s 138s 140s 142s 144s 146s 148s 150s 160s 162s 164s 166s 168s 170s 172s 174s 176s 178s 180s 182s 184s PM CAPRI-C2 PM CAPRI-PH PM CAPRI-R472A PM CAPRI-WT 46s 48s 50s 52s 54s 56s 58s 60s 62s 64s 66s 64s 62s 60s 58s 54s56s 50s52s 48s 46s 24s 26s 28s 30s 32s 34s 36s 38s 40s 42s 44s 42s 38s40s 36s 34s 32s 30s 28s 26s 24s 24s 26s 28s 30s 32s 34s 36s 38s 40s 42s 44s 42s 38s40s 36s 34s 32s 30s 28s 26s 24s 24s 26s 28s 30s 32s 34s 36s 38s 40s 42s 44s 42s 38s40s 36s 34s 32s 30s 28s 26s 24s 0s 4s 6s 8s 10s 12s 14s 16s 18s 20s 22s 20s 18s 16s 14s 12s 10s 8s 6s 4s 0s 22s 20s 18s 16s 14s 12s 10s 8s 6s 4s 0s 24s 26s 28s 30s 32s 34s 36s 38s 40s 42s 44s 42s 38s40s 36s 34s 32s 30s 28s 26s 24s 0s 4s 6s 8s 10s 12s 14s 16s 18s 20s 22s 20s 18s 16s 14s 12s 10s 8s 6s 4s 0s 0s 4s 6s 8s 10s 12s 14s 16s 18s 20s 22s 20s 18s 16s 14s 12s 10s 8s 6s 4s 0s Xu et al., Figure S9 etal., Xu A B Total path length (mm) Directionality 100 150 200 250 300 0.2 0.4 0.6 0.8 1.2 1.4 50 IL-8 gradient 0 0 1 NG 0.1 nM 1 nM 100 nM 1 mM NG CTL NG CTL kd caprikdcapri p caprikdcaprikd p = 0.003 0.1 nM 0.1 0.1 nM 0.1 CTL CTL = 0.01 caprikdcaprikd caprikdcaprikd 1 nM CTL 1 nM CTL caprikdcaprikd caprikdcaprikd 100 nM100 CTL 100 nM100 CTL CTL kd kd p kaprikdcapri p kaprikdcapri = 0.002 = 0.001 1 1 uM 1 1 uM m CTL CTL m M M kd kaprikdcaprikd kaprikdcapri Roundness (%) 100 120 140 Speed (mm/min) 10 15 20 25 20 40 60 80 0 5 0 NG NG CTL CTL kd kd caprikdcapri caprikdcapri p p = 0.003 0.1 nM 0.1 0.1 nM 0.1 = 0.001 CTL CTL capri caprikdcaprikd caprikdcaprikd kd 1 nM 1 nM CTL CTL caprikdcaprikd caprikdcaprikd 100 nM 100 100 nM100 CTL CTL Figure S10 etal., Xu kd kd p kaprikdcapri p kaprikdcapri = 0.001 = 0.006 1 1 1 uM 1 uM m CTL m CTL M M kaprikdcaprikd kaprikdcaprikd Supplemental Information Supplemental Figure Legends: Fig. S1. GPCR-mediated chemotaxis of HL60 cells in response to fMLP and IL8 gradients with an enormous concentration range. (A) Montages show the travel path of chemotaxing HL60 cells in response to a wide range of fMLP and IL-8 gradients. (B) Chemotaxis behaviors measured from A and described as three parameters: directionality, where 0 represents random movement and 1 represents straight movement toward the gradient; speed, defined as the distance that a cell’s centroid moves as a function of time; total path length, the total distance a cell has traveled. Fig. S2. Mammalian neutrophils and HL60 cells highly express CAPRI. (A) The expression pattern of RasGAP proteins in human and mouse neutrophils. (B) The expression pattern of RasGAP proteins in human neutrophil-like (HL60) cells during DMSO-induced differentiation. Aliquots of cells, which were differentiated in the RPMI culture media with 1.3 % DMSO for the indicated days as described in the session of Materials and Methods, were mixed with SDS loading buffer, sonicated, and then subject to western blot detection of interested proteins of interests with their specific antibodies. Fig. S3. Three plasma membrane markers colocalize in HL60 cells before and after chemoattractant fMLP stimulation. (A) Colocalization of two membrane markers (C1AC1A- YFP and CAAX-mCherry) in HL60 cells. Cell expressing two plasma membrane (PM) markers, C1AC1A-YFP (green) and CAAX-mCherry (red), was stimulated with fMLP at time 0s. During the period of imaging, C1AC1A-YFP colocalizes with in the cells before and after fMLP stimulation. (B) Colocalization of two membrane markers (mem-cerulean and CAAX-mCherry) in HL60 cells. Cell expressing two plasma membrane (PM) markers, mem-cerulean (cyan) and CAAX-mCherry (red), was stimulated with fMLP at time 0 s. Mem-cerulean colocalizes with CAAX-mCherry before and after fMLP stimulation. Scale bar = 10 m in A and B. Fig. S4. caprikd/OE cell displayed little membrane translocation of active Ras probe RBD-RFP while displayed a strong membrane translocation of CAPRI. The caprikd/OE cell expressing PM marker (cyan), CAPRI-tGFP (green), and active Ras probe RBD-RFP (red) was stimulated with 1 M fMLP at time 0 s and displayed a clear membrane translocation of CAPRI-tGFP and little membrane translocation of RBD-RFP. Scale bar = 10 m.

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