Rabbit anti jak2

Unless otherwise stated, all chemicals were obtained from the Sigma Chemical Company, St. Louis, MO, USA. Mouse monoclonal anti-mouse TfR1 (cat.13-6800) was purchased from Invitrogen, Carlsbad, CA, USA; rabbit polyclonal anti-IRP1 (cat. ab236773), rabbit polyclonal anti-Fpn1 (cat. ab78066) and anti-DMT1 (cat. ab123085) from Abcam, Inc., Cambridge, UK; rabbit polyclonal anti-FTL (cat. 10727-1-AP) from Protein-tech, Chicago, IL, USA; mouse anti-p-JAK2 (cat. 3771), rabbit anti-JAK2 (cat. 3230), mouse anti-p-STAT3 (cat. 9145) and rabbit anti-STAT3 (Tyr705) (cat. 9139) from Cell Signaling Technology, Boston, USA; goat anti-rabbit or anti-mouse IRDye 800 CW secondary antibody (cat. 926-32210) from LI-COR Bio Sciences, Lincoln, NE, USA; FastStart Universal SYBR Green Master (cat. 11123) and AevertAid First Strand cDNA Synthesis Kit (cat. 11119ES60) from Yeasen BioTechnologies Co., Shanghai, China; LightCycler96 (cat. 05815916001) from Roche, Nutley, NJ, USA; TRIzol reagent (cat.15596018) from Life Technologies, Carlsbad, CA, USA; BCA protein Assay kit (cat. 23225) from Thermo Scientific, Waltham, MA, USA; and protein RIPA lysis buffer (cat. P0013B) from Beyotime Institute of Biotechnology, Haimen, JS, China.

The cells in each group were lysed in M-PER cell lysis buffer (Thermo Fisher Scientific, USA). The total protein of each group was collected and quantified measuring with a BCA total protein kit (Thermo Fisher Scientific, USA). Proteins of the same volume were separated by SDS-PAGE. Then, the proteins were transferred to the semidry membrane. Subsequently, membranes were blocked in 5% BSA for 1 hour at room temperature and incubated with β-actin (1 : 2500, Proteintech), rabbit anti-BAX (1 : 250, Proteintech), rabbit anti-Bcl-2 (1 : 1000, Abcam), rabbit anti-JAK2 (1 : 1000, Cell Signaling Technology), and rabbit anti-STAT3 (1 : 1000, Cell Signaling Technology) antibodies at 4°C overnight. Subsequently, the membranes were incubated with secondary antibodies labeled with horseradish peroxidase (HRP) for 2 hour. The membrane was exposed to the developing solution of electrogenerated chemiluminescence (ECL) (Advansta, CA, USA). The optical density of protein bands was analyzed by ImageJ image analysis software.

Western blotting was performed using a standard method described previously [12 (link)]. The nitrocellulose membranes were incubated with following antibodies: mouse anti-BAX (Sigma-Aldrich, St. Louis, MO, USA, Cat# WH0000581M1, RRID:AB_1840183,), mouse anti-BCL-2 (Sigma-Aldrich Cat# B3170, RRID:AB_258541), rabbit anti-phospho Akt1/2/3 (Santa Cruz Biotechnology, Dallas, TX, USA, Cat# sc-7985 also sc-7985-R, RRID:AB_667741), rabbit anti-Akt1/2/3 (Santa Cruz Biotechnology, Cat# sc-8312, RRID:AB_671714), rabbit anti-phospho-MAPK (Thermo Fisher Scientific, Waltham, MA, USA, Cat# PA1-14302, RRID:AB_1086514), rabbit anti-MAPK (Thermo Fisher Scientific Cat# PA5-14425, RRID:AB_2141578), rabbit anti-pospho JAK2 (Cell Signaling Technology, Leiden, The Netherlands, Cat# 3771S, RRID:AB_330403), rabbit anti-JAK2 (Cell Signaling Technology Cat# 4040S, RRID:AB_10691469), and mouse anti- β-Actin (Sigma-Aldrich Cat# A5316, RRID:AB_476743).

Tissues were homogenized in RIPA lysis buffer with protease inhibitor. The homogenate was centrifuged at 12,000 rpm for 30 min at 4 °C. Protein concentration was assayed with the BCA method. Protein was loaded and subjected to electrophoresis in 10% SDS-PAGE gels and transferred onto PVDF membranes. The membranes were then blocked in 5% BSA for 2 hour. The membranes were incubated with primary antibodies as follows: rabbit anti-JAK2 (1:1000, Cell Signaling Technology), or rabbit anti-p-JAK2 (1:1000, Cell Signaling Technology), or mouse anti-STAT3 (1:1000, Cell Signaling Technology), or mouse anti-p-STAT3 (1:2000, Cell Signaling Technology), or rabbit anti-β-actin (1:6000, Abcam), or rabbit anti-VEGF (1:1000, Servicebio), or rabbit anti-bFGF (1:1000, Bioworld), or rabbit anti-angiostatin (1:1000, Bioworld), or rabbit anti- endostatin (1:1000, Boster), with gentle shaking at 4 °C overnight. Then, horseradish peroxidaseconjugated goat anti-mouse IgG (1:50000, Proteintech) or goat anti-rabbit IgG (1:50000, Proteintech) secondary antibodies were incubated with the membranes for 2 hours at room temperature. The immunopositive bands were visualized using an enhanced chemiluminescent substrate (Thermo Fisher) and Bio-Rad ChemiDoc XRS digital documentation system. The amount of protein expression is presented relative to the levels of β-actin.

The L4-L5 segments of the spinal cord were homogenized and subjected to SDS-PAGE as previously described48 (link). The membranes were incubated with the following primary antibodies at 4 °C overnight: goat anti-IL-1β (1:500; R&D), rabbit anti-caspase-1 (1:500; Abcam), rabbit anti-NALP1 (1:500; Abcam), rabbit anti-ASC (1:500; sc-22514-R; Santa Cruz Biotechnology, Santa Cruz, CA, USA), goat anti-mIL-1β (1:500; sc-23460; Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-pSTAT3 (1:500; Cell Signaling Technology), rabbit anti-STAT3 (1:1000; 79D7; Cell Signaling Technology, USA), rabbit anti-pJAK2 (1:500; 3771; Cell Signaling Technology, USA), rabbit anti-JAK2 (1:1000; 3230; Cell Signaling Technology, USA), and mouse anti-β-actin (1:5000; 4967; Cell Signaling Technology, USA). The blots were then washed in TBST and incubated in the appropriate secondary antibody for 1 hour at room temperature. The secondary antibodies were donkey anti-goat lgG-HRP (1:10000; sc-2020; Santa Cruz Biotechnology, CA, USA), donkey anti-mouse lgG-HRP (1:5000; sc-2314; Santa Cruz Biotechnology, CA, USA), or HRP Affinipure Goat Anti-Rabbit lgG (H+L) (1:10000; E030120-01; ErthOx, CA, USA). The western blot images were captured on an ImageQuant LAS4000 mini image analyser (GE Healthcare, Buckinghamshire, UK), and the band levels were quantified using the Image J software, version 1.42q.

Proteins from samples (n = 4/group) were extracted in RIPA lysis buffer (Solarbio, China) with PMSF and a phosphatase inhibitor, and the protein concentration was assessed by BCA protein assay kit (Solarbio, China). Proteins were separated on a 7.5% sodium dodecyl sulfate-polyacrylamide electrophoresis gel (SDS-PAGE) and then transferred onto polyvinylidene fluoride membranes. The membranes were blocked with 5% non-fat milk in TBST for 1 h and incubated overnight at 4°C in primary antibodies: rabbit anti-JAK2 (1:2,000; Cell Signaling Technology, United States), rabbit anti-phospho-JAK2 (Y1007 + 1,008) (1:2,000; Abcam, United States), rabbit anti-STAT3 (1:2,000; Cell Signaling Technology, United States), rabbit anti-phospho-STAT3 (Tyr705) (1:2,000; Cell Signaling Technology, United States), rabbit anti-β-actin (1:4,000; Proteintech Group, China). Later, the membranes were washed in TBST and then cultured with anti-rabbit HRP-conjugated secondary antibody (1:4,000; Proteintech Group, China) for 1 h. Finally, the enhanced chemiluminescence assay kit (Proteintech Group, China) was used to visualize immunoblots, and the densities of the relative target proteins were measured using ImageJ. The β-actin was chosen as the internal reference control.

The Jak2 specific inhibitor, AG490 (Sigma-Aldrich), was dissolved in ethanol at a concentration of 5 mM and stored at −20 °C. The working concentration of AG490 in culture medium is 5 μM. Cell lysates were collected by using RIPA buffer containing proteinase and phosphatase inhibitors. The proteins were separated by SDS-PAGE (Sigma-Aldrich) and transferred to a PVDF membrane (Millipore). After blocking by 5% non-fat milk in TBST, the membrane was incubated with appropriately diluted primary antibodies. Rabbit anti-Jak2 (Cell Signaling Technology, Danvers, MA, USA), mouse anti-phospho-Jak2 (Tyr1007/1008) (Cell Signaling), rabbit anti-Stat3 (BD Pharmingen, San Diego, CA, USA), rabbit anti-phospho-Stat3 (Ser727) (Cell Signaling), rabbit anti-EGFR (GeneTex, Irvine, CA, USA), rabbit anti-phospho-EGFR (Tyr1068) (GeneTex), rabbit anti-ERK (GeneTex), rabbit anti-phospho-ERK (Thr202/Tyr2004) antibodies (Invitrogen), rabbit anti-Sox2 (GeneTex), rabbit anti-Oct4 (GeneTex), rabbit anti-Nanog (Biorbyt) and mouse anti-beta-actin (Millipore) were used in this research. The enhanced chemilumescent (ECL) western blotting substrate kit (Thermo) was used to stain the membranes and signals were obtained using UVP BioSpectrum Imaging System (Upland, CA, USA).