Rabbit anti adam17

The following antibodies were used at the indicated dilutions: c‐Myc (9E10, sc‐40, 1:1,000, Santa Cruz Biotechnology), Separase (A302‐215A, 1:2,000, Bethyl Laboratories), KIF4A (A301‐074A, 1:1,000, Bethyl Laboratories), Axin1 (#2087, 1:1,000, Cell Signaling Technology), B56α (610615, 1:3,000, BD Biosciences), BubR1 (A300‐995A,1:1,000, Bethyl Laboratories), PP2A catalytic subunit (05‐421, 1:2,000, Millipore), PP2A scaffold subunit (#2041, 1:1,000, Cell Signaling Technology) GFP (ab290, 1:4,000, Abcam), FoxO3 pS413 (#8174, 1:1,000, Cell Signaling Technology), FoxO3 rabbit polyclonal α‐pS253 (Raised against peptide CAPRRRAV(pS)MDNS; 1:500, Moravian Biotechnology), Anti‐RFP (1:1,000; MBL, FM005), Anti‐GFP (1:1,000; Roche, 11814460001), Rabbit anti‐ADAM17 (1:1,000, Abcam, 2051), Rabbit anti‐ADAM17 (1:1,000, Abcam, 39162), Rabbit anti‐EGFR (1:1,000, cell signaling, 2232), Rabbit anti‐pEGFR Y1068 (1:1,000, cell signaling, 2234), Mouse anti‐Transferrin receptor (1:1,000, Invitrogen, 136800), Mouse anti‐GAPDH (1:5,000, Sigma, G8795), rabbit Anti‐GFP (1:3,000, Takara Bio Clontech, 632592), Donkey anti‐rabbit‐HRP (1:2,000, GE Healthcare, NA934), Sheep anti‐mouse‐HRP (1:2,000, GE Healthcare, NXA931), and H3pS10 (06‐570, 1:1,000, Millipore).

Mouse B220+ B cells or human PBMC were blocked with 10 μg 2.4G2 or human FcR blocker (Miltenyi Biotec), respectively. Human PBMC was stained with anti-human PE-CD19, APC-CD14, or PE-CD3 (Biolegend). Mouse and human cells were washed, fixed, permeabilized, blocked again, and stained with their respective antibodies: FITC-hADAM10 (R&D), PE-mADAM10 (R&D) and/or rabbit-anti-ADAM17 (Abcam) followed by DyLight-649 anti-rabbit IgG (Biolegend). Isotype control for hADAM10 is mouse IgG2b FITC (R&D), for mADAM10 is Rat IgG2a PE (R&D), and for ADAM17 is rabbit IgG. Tyramide Signal Amplification (Kit #26) was used for mouse B cell TNF staining as described [8 (link)]. Dead cells and aggregates were excluded by scatter gating, examined on a BD Canto, and analyzed with FCS Express, v.4.

The cells were lysed in the suitable buffer, and the lysates were centrifuged at 13,000 rpm for 20 min at 4°C. The protein concentration was measured using a BCA kit (Beyotime Biotechnology, Shanghai, China). Equal amounts of protein (10 μg) from each sample were boiled in the loading buffer for 5 min and resolved by 12% SDS-PAGE. The protein bands were transferred onto an NC membrane (0.45 μm pore size; Millipore, USA) that was blocked in 3% BSA-TBST for 30 min, and then incubated overnight with mouse anti-Beclin-1 (1 : 2000; Immunoway, USA), rabbit anti-LC3-II (1 : 2000; Immunoway, USA), mouse anti-BACE1 (1 : 1000; Immunoway, USA), rabbit anti-PS-1 (1 : 2000; Immunoway, USA), and rabbit anti-ADAM17 (1 : 1000; Abcam, UK) primary antibodies at 4°C. After washing 5 times with TBST, the membrane was incubated with HRP-conjugated goat anti-rabbit IgG (H + L) or goat anti-mouse IgG (H + L) (1 : 10000; Beyotime, Shanghai, China) secondary antibodies for 40 min at room temperature. The blots were washed again, developed using an ECL reagent (Millipore, USA) for 5 min, and the film was exposed and photographed. GAPDH was used as the internal control. The bands were scanned, and the relative gray values were calculated using ImageJ software.