Anti btk

Following are the steps for immunohistochemistry: Deparaffinization of the paraffin sections of renal tissues followed by hydration, blocking using an endogenous peroxidase, antigen retrieval, incubation in xeno- and serum-free medium, addition of primary antibody (4 °C, overnight), and then incubation with secondary antibody (37 °C, 30 min). Finally, DAB was used as chromogen and hematoxylin was used as counterstain. The primary antibodies used are as follows: anti-BTK (1:100, Cell Signaling, MA, USA), anti-pBTK (1:100, Affinity Biosciences, Cincinnati, USA), anti-CD68 (1:200, Arigo, Taiwan, China), p-NF-κB p65 (1:100, Affinity, Cincinnati, USA), TNF-ɑ (1:500, Arigo, Taiwan, China), IL-1β (1:100, Affinity, Cincinnati, USA), C3b (1:100, Abcam, MA, USA), and C4d (1:100, Abcam, MA, USA). The secondary antibody used is PV-6000 (Zsbio, Beijing, China). Sections of renal tissues incubated in PBS were used as negative controls. The Image-Pro-Plus software version 6.0 was used to analyze the immunohistochemical staining.

CD95L treated (40 ng/ml) or non-treated cells were washed with PBS containing phosphatase inhibitors, pelleted, and lysed on ice for 30 min with Pierce IP Lysis buffer (Fisher Scientific, Germany) containing vanadate, inhibitors for phosphatase and proteinase. Lysates of 500 μg protein were immunoprecipitated at 4°C for 4 hr with the anti-mouse CD11a (M17/4, biolegend), anti-mouse Btk (Cell Signaling, USA), anti-mouse CD11b (M1/70, ebioscience, USA) antibodies or the corresponding isotype controls. Afterward, 40 μl Dynabeads M-280 Streptavidin was added to each sample and incubated for 1 hr at 4°C with rotation. Beads were washed 5 times with 1 ml of lysis buffer. The immunoprecipitates were released by cooking the beads with 40 μl of 2x laemmli buffer at 95°C for 5 min.
Immunoblotting was performed as previously described (Letellier et al., 2010 (link)). Membranes were probed with following antibodies respectively: anti-phospho-Syk (Tyr319, 352), anti-phospho-Btk (Tyr223), anti-phospho-PLCγ2 (Tyr1217), anti-Syk, anti-Btk, anti-PLCγ2 (Cell Signaling, USA), anti-Rap1 (Fisher Scientific), anti-mouse CD11a, anti-CD95 (M20, Santa Cruz Biotechnology, Germany), anti-mouse CD11b (Novus Biologicals, USA). Western blots were quantified with ImageJ software and normalized to the respective loading controls.

Ibrutinib was purchased from LC Labs. Cobicistat and ketoconazole were purchased from MedChemExpress. Reference standards of Ibrutinib, its primary metabolite PCI-45227 (M37) and their corresponding deuterated versions, used as internal standards for the analytic method, were purchased from AlsaChim. PEG300, PEG400, corn oil, and polysorbate-80 (Tween-80) were purchased from Sigma-Aldrich, and LC-MS–grade formic acid (FA), methanol, and acetonitrile were from Thermo Fisher Scientific. Blank plasma was obtained from FVB wild-type mice (Taconic Biosciences). The following antibodies were obtained from Cell Signaling Technology: anti-BTK (catalog no. 8547). Anti-phospho-BTK (Y223, catalog no. ab68217) was purchased from Abcam and anti-GAPDH (catalog no. MAB374) was purchased from Millipore Sigma.

PBMCs were resolved in lysis buffer with a protease inhibitor (Beyotime, Shanghai, China) at 4 °C for 30 min. They were then centrifuged at 12,500 rpm and 4 °C for 20 min and the supernatant was collected after centrifugation. Protein concentration was measured using Enhanced BCA Protein Assay Kit (Beyotime, Shanghai, China). Next, the protein was added to loading buffer and the mixture was boiled in boiling water for 10 min. The total protein was electro-transferred to PVDF membranes. The membranes were successively incubated with primary antibody and secondary antibody combined with horseradish peroxidase. The protein bands were observed using a chemiluminescence system. The primary antibodies used are as follows: anti-BTK, anti-pBTK (Cell Signaling, MA, USA), NF-κB p65 (Cell Signaling, MA, USA), phospho-NF-κB p65.

Antibodies purchased from Cell Signaling Technology (CST, Danvers, MA) including anti-FLT3 (Cat.#3462), anti-phospho-FLT3 Tyr589/591 (Cat.#3464), anti-STAT5 (Cat.#9363), anti-phospho-STAT5 (Cat.#9351), anti-ERK (Cat.#4695), anti-phospho-ERK (Cat.#4377), anti-Src family kinase (Cat.#2109), anti-phospho-Src family kinases Tyr416 (Cat.#2101), anti-SYK (Cat.#13198), anti-phospho-SYK Tyr525/526 (Cat.#2711), anti-BTK (Cat.#8547), anti-phospho-BTK Tyr223 (Cat.# 87457), anti-MCL1 (Cat.# 94296), anti-BCLXL (Cat.# 2764), and anti-HSP90 (Cat.#4874). Anti-β-Actin (Cat.# SC-1616) was purchased from Santa Cruz Biotechnology (Dallas, TX) and anti-GAPDH (Cat.# MAB374) from EMD Millipore (Burlington, MA). HSP90, β-Actin, and GAPDH were used as loading controls.