Goat anti human β actin

(Z‐Leu‐Leu‐NHCH2)2CO, 1,3‐di‐(N‐carboxybenzoyl‐ l‐leucyl‐ l‐leucyl)amino acetone, and 2,2′‐(2‐oxo‐1,3‐propanediyl)bis[N‐[(phenylmethoxy)carbonyl]‐ l‐leucyl‐ l‐leucinamide) ((Z‐LL₂)‐ketone) were purchased from Santa Cruz (sc‐311559); N‐morpholinurea–leucine–homophenylalanine–vinylsulfone‐phenol (LHVS) was synthesized in the laboratory of Dr. C Driessen, Department of Oncology and Hematology, Cantonal Hospital St Gallen, St Gallen, Switzerland, following protocols as published in [27]. Recombinant IFN‐γ was purchased from eBioscience (34‐8319‐82). The following antibodies were used: mouse anti‐human LAMP‐1 PE (BD; clone H4A3; 555801), mouse anti‐human CLIP FITC (BD; clone CerCLIP; 555981) mouse anti‐human CD74 PE (Biolegend; clone LN2; 326807), mouse anti‐human CD14 Pacific blue (Biolegend; clone M5E2; 301815), mouse anti‐human HLA ABC PE‐Cy7 (BD; clone G46‐2.6; 561349), mouse anti‐human HLA DR PerCP (Biolegend; clone L243; 560896), mouse anti‐human CD74 N‐terminal (Abcam; clone 2D1B3; ab181465), goat anti‐human β actin (Santa Cruz; clone I‐19; SC1616), goat anti‐mouse AF647 (Thermofisher Scientific; A28181), rabbit anti‐human Na‐K ATPase (Cell Signalling; 3010s), mouse anti‐human LAMP1 (BD, clone H4A3; 555798), Rabbit Anti‐mouse‐HRP antibody, (Dako; P0161), and Rabbit anti‐goat HRP antibody (Dako; P0160).

Cells were lysed with RIPA lysis buffer containing protease inhibitors for 1 h on ice. The cells were then centrifuged at 14,500 rpm for 15 min, the pellet was discarded, and the supernatant was collected. Electrophoresis was performed under denaturating conditions on 10% SDS polyacrylamide gel. The proteins were transferred to a nitrocellulose membrane. Blots were saturated in Odyssey buffer/PBS (1:1) for 1 h at RT. The blots were incubated with primary antibodies Rabbit anti-human P2X7R (Alomone, Jerusalem BioPark (JBP), Israel), or Rabbit anti-human P2Y12R (Ananspec, LIEGE Science Park, Belgium) in Odyssey buffer/PBS (1:1) + Tween-20 (0.1%) overnight at 4 °C followed by fluorescently labeled secondary antibody (Odyssey) in Odyssey buffer/PBS (1:1) + Tween-20 (0.1%) for 1 h at room temperature. Blots were imaged using Odyssey imager. Blot were then washed and stained with goat anti-human β-actin (Santa Cruz, Heidelberg, Germany) antibody following the same procedure as above. Western blot relative quantification was realized by calculating the ratio of the integrated intensity of the band of the protein of interest over the integrated intensity of the band of β-actin.

Western blotting was performed as described in [13 (link)]. Purified neutrophils from GPA patients and HCs were resuspended in Laemmli buffer and 2 x10⁵ cells analyzed on a 4–12% NuPage Bis-Tris gradient gel (Invitrogen). Antibodies: Rabbit anti-human proteinase 3 (1:250; Abcam 103632) succeeded by HRP-conjugated goat-anti-rabbit (1:1000; DAKO P0448). The membrane was stripped and reprobed with goat-anti-human-β-actin (1:500; Santa Cruz sc-1616) succeeded by HRP-conjugated rabbit-anti-goat (1:1000; DAKO P0449). The membrane was developed by chemiluminiscence using SuperSignal West Pico (Pierce) and analyzed on a Bio-Rad Chemidoc (Bio-Rad). Quantification of band intensities was performed with ImageLab software (Bio-Rad).