Anti phospho erk1 2

Western blot analysis of cell extracts was performed as described previously (Ohnishi et al. 2017 (link)). Briefly, cells were washed with phosphate-buffered saline three times then lysed in RIPA buffer [50 mmol/L Tris-HCl (pH 8.0), 150 mmol/L NaCl, 0.5% (w/v) sodium deoxycholate, 0.1% (w/v) sodium dodecyl sulfate, and 1.0% (w/v) NP-40 substitute]. After incubation on ice for 20 min, debris was removed by centrifuging at 2000 g for 10 min at 4 °C. Supernatant proteins (20 μg) were electrophoresed with sodium dodecyl sulfate polyacrylamide gel and transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). Nonspecific binding was blocked by incubating membranes with Blocking One–P (Nacalai Tesque, Kyoto, Japan) for 20 min at room temperature. Then membranes were incubated overnight with primary antibodies, including anti-phospho-Smad1/5/8 (1:1000), anti-Smad1 (1:1000), anti-phospho-ERK1/2 (1:1000), and anti-ERK1/2 (1:1000), anti-phospho-p38 (1:1000), anti-p38 (1:1000) [all from Cell Signaling Technology, Danvers, MA, USA], at 4 °C and then with secondary antibodies for 45 min at room temperature. Immunoreactive protein bands were visualized using an ECL-peroxidase detection system (Thermo Fisher Scientific) and LAS-4000 (Fujifilm, Tokyo, Japan).

Immunoblotting was carried out as described previously [11 (link)]. The following antibodies were purchased from Abcam: anti-GAPDH (1:2500, 37 kDa, AB9485, RRID:AB_307275), anti-IL-6 (1:2000, 17 and 50 kDa, ab9324, RRID:AB_307175), and anti-ICAM-1 (1:4000, 90 kDa, AB53013, RRID:AB_870702). Anti-ACE2 (1:1000, 125 kDa, MA532307, RRID:AB_2809589) and anti-HIF-1α (1:1000, 130 kDa, PA5-85,494, RRID:AB_2792634) antibodies were obtained from Thermo Scientific. The following antibodies were purchased from Cell Signaling Technology: anti-ERK1/2 (1:1000, 42, 44 kDa, #4695, RRID:AB_390779), anti-phospho-ERK1/2 (Thr202/Tyr204), (1:1000, 42, 44 kDa, # 9101, RRID:AB_331646), anti-MSK1 (1:1000, 95 kDa, #3489, RRID:AB_2285349), anti-phospho-MSK1 (Thr581) (1:1000, 95 kDa, #9595, RRID:AB_2181783), anti-p38 (1:1000, 43 kDa, #9212, RRID:AB_330713), anti-phospho-p38 (Thr180/Tyr182) (1:1000, 43 kDa, #4511, RRID:AB_2139682), anti-phospho-JNK (Thr183/Tyr185) (1:1000, 46, 54 kDa, #4668, RRID:AB_823588), anti-JNK (1:1000, 46, 64 kDa, #9252, RRID:AB_2250373), anti-phospho-NFκB p65 (Ser536) (1:1000, 70 kDa, #3033, RRID:AB_331284), and anti-NFκB p65. (1:1000, 70 kDa, #6956, RRID:AB_10828935). The following antibody were obtained from ABclonal (Woburn, MA): anti-phospho-NFkB p65 (S276) (1:1000, 70 kDa, AP0123, RRID:AB_2771505). An anti-phospho-MBP antibody (1:200, 18 kDa, 13–104) was purchased from Merck.

For stimulation, WSU-FSCCL cells were incubated with antibodies and/or DC-SIGN-Fc/HA at 4 °C for 30 min and then rapidly warmed to 37 °C to initiate signaling for the time indicated. Alternatively, FL cells were pre-incubated with DC-SIGN-HA at 4 °C prior to anti-IgM stimulation and rapidly warmed to 37 °C to initiate signaling for 30 s. SDS-PAGE was performed as previously described^{6 (link)} using the following primary antibodies: anti-phospho-ERK1/2, anti-ERK1/2, anti-phospho-AKT^(S473), anti-AKT, anti-phospho-SYK^(S525,526), anti-SYK (all from Cell Signaling Technology), anti-phospho-LYN^(Y396) (Abcam), anti-LYN (Santa Cruz Biotechnologies), anti-HSC70 (Santa Cruz Biotechnologies) and anti-GAPDH (Invitrogen). Images were captured using the ChemiDoc-It Imaging System with a BioChemi HR camera (UVP) and quantified using ImageJ (http://imagej.nih.gov/ij/).

Antibodies were obtained from the indicated supplier: anti-PP2A A subunit (Santa Cruz Biotech, CA, USA, sc-6112), anti-PP4c (Bethyl, TX, USA), anti-phospho ERK1/2, anti-phospho Thr308 Akt, anti-total ERK1/2, anti-total Akt (Cell Signaling, MA, USA), anti-FLAG tag (Sigma, MO, USA), anti-ubiquitin (Life Sensors, PA, USA), anti-PME-1 (LifeSpan BioScience, WA, USA), anti-demethyl PP2Ac (Merck Millipore, MA, USA, 05–577), anti-total PP2Ac (Millipore, 07–324), anti-tubulin alpha (Thermo Scientific, MA, USA), p97/VCP (GeneTex, CA, USA). Anti-PP6c was generated as previously described [20 (link)].

Anti-pan Akt (#2920), anti-phospho S473 Akt (#4058), anti-ERK1/2 (#9102), anti-phospho ERK1/2 (#9106) were from Cell Signalling (Danvers, Massachusetts, USA). Anti-Sx4 (#110042) and Anti-SNAP23 (#111202) were from Synaptic Systems (Goettingen, Germany). Anti-GLUT1 (#652) and anti-GLUT4 (654) were from AbCam (Cambridge, United Kingdom) and anti-GAPDH (#4300) was from Ambion (Foster city, California, USA). Detection antibodies were from LI-COR Biosciences (Lincoln, Nebraska, USA).