β actin sc 81178

Protein extraction from dorsal skin tissues was carried out using a PRO-PREP (Intron Biotechnology, Seoul, Republic of Korea) with a protease and phosphatase inhibitor cocktail (Sigma Aldrich In., St Louis, MO, USA). The extracted protein concentration was determined by Bradford’s assay and bovine serum albumin (BSA) was used as a standard quantification. Proteins were separated with 8–10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and transferred to a polyvinylidene difluoride (PVDF) membrane. The membrane was reacted for 12 h at 4 °C with primary antibodies in 5% skim milk, and then reacted with secondary antibodies in 5% skim milk for 2 h at 20 to 25 °C. Bands were detected by enhanced chemiluminescence (ECL) substrate and visualization was performed using an ImageQuant^™ LAS-4000 (FUJIFILM, Tokyo, Japan). Antibodies of pro-COL1A1 (sc-25973), ERK (sc-93), JNK (sc-7345), c-Fos (sc-253), and β-actin (sc-81178) were obtained from Santa Cruz Biotechnology Inc. (Dallas, TX, USA). p-ERK (#4377), p-JNK (#4668), p-p38 (#9215), p-38 (#9212), p-c-Fos (#5348), TGF-β (#3711), p-Smad 2/3 (#8828), and Smad 2/3 (#5678) were purchased from Cell Signaling Technology Inc. (Danvers, MA, USA).

Reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless stated otherwise. Antibodies for p38α MAPK: sc-535; p-p38α MAPK: sc-166182; NF-κB: sc-56735; β-actin: sc-81178, were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Opti-4CN substrate kit was acquired from Bio-Rad Laboratories (Hercules, CA, USA). Synthetic substrates for thrombin (S-2238™, H-D-Phe-Pip-Arg-pNa), factor XIa (S-2366, pyroGlu-Pro-Arg-pNa), plasma kallikrein and factor XIIa (S-2302™, H-D-Pro-Phe-Arg-pNA), factor Xa (S-2222™, Bz-Ile-Glu-Gly-Arg-pNa) and factor VIIa (SCP-0248, MeSO2-Cha-Abu-Arg-pNA)) were purchased from Chromogenix (Milan, Italy). Aprotinin (Trasylol^®) was purchased from Bayer (São Paulo, Brazil). Ellagic acid, rabbit brain thromboplastin, phospholipids were purchased from Wiener Lab (Rosario, Argentina).

MAP2 (A-4, sc-74421), MBP2 (10458-1-AP, Proteintech, A-3, sc-376995), β-actin (sc-81178) and Ubiquitin (Ub) (P4D1, sc-8017) were from Santa Cruz Biotechnology, Inc. Postsynaptic density 95 (PSD95) (ab18258), and synaptophysin (ab8049) were from Abcam. NeuN (MAB377) was from Millipore Biotechnology. Anti-PHF1 primary antibody is a gift kindly from Dr. Peter Davies, Albert Einstein College of Medicine. Alexa-conjugated secondary antibodies were from Molecular Probes/Invitrogen (Carlsbad, CA). TUNEL (Terminal deoxynucleotidyl transferase-mediated Dutp nick end labeling) was carried out using the ‘In Situ” Cell Death Detection Kit, Fluorescein’ kit (Cat #11684795910, Roche Molecular Biochemicals. Golgistaining kit (FD Rapid GolgiStain ^TM Kit) was from FD Neurotechnologies. INC. Amyloid beta1–42 ELISA kit (KMB3441) was from Thermo Fisher Scientific Inc. Polyvinylidene difluoride (PVDF) membranes with pore size of 0.45 or 0.2 μm were from Millipore Biotechnology (USA). BCIP (5-bromo-4-chloro-3-indolyl-phosphate) and NBT (nitro blue tetrazolium) were from Promega (Madison, WI). All the other chemicals were from Sigma-Aldrich (St. Louis, MO) unless indicated otherwise.

Protein extracts were isolated from the colon tissues (30 mg) using the Pro-prep™ (Intron biotechnology Inc., Gyeonggi-do, Korea). The extracted protein samples (30 μg) were separated on an 8–12% sodium dodecyl sulfate–polyacrylamide gel and transferred to an immobilon-P PVDF membrane (IPVH00010, Millipore, MA, USA), as described previously [25 (link)]. The membrane was incubated with 2.5% skimmed milk for 30 min at 22–25 °C and then probed overnight with a primary antibody (dilution 1:1000 in Tween 20/Tris-buffered saline) at 4 ℃. Primary antibodies against Bcl-2 (sc-7382), Cdk4 (sc-23896), IκB-α (sc-203), STAT3 (sc-482), XIAP (sc-55552), and β-actin (sc-81178) were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). The p65 (#4764), pIκB-α (#9246), p-STAT3 Tyr705 (#9145), and p-STAT3 Ser727 (#9134) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). After washing three times with Tween 20/Tris-buffered saline, the membrane was incubated with horseradish peroxidase-conjugated secondary antibody (Jackson ImmunoResearch, West Grove, PA, USA, dilution, 1:2000) for 2 h at 22–25 °C. The blots were visualized using enhanced chemiluminescence (Ab signal, Seoul, Korea).

All antibodies applied were commercially purchased: mouse monoclonal antibody to β-Amyloid, 17–24 (4G8) (SIG-39245, Covance, CA, USA); rabbit polyclonal antibody A11 to Aβ oligomers (Error! Hyperlink reference not valid., Invitrogen, CA, USA); mouse monoclonal antibody to pathological tau, AT-8(pSer202/Thr205) (MN1020, Thermo Scientific, IL, USA); rabbit antibody to synaptophysin, a presynaptic marker (Sigma Aldrich, USA); rabbit polyclonal antibody to Iba-1, a marker of activated microglia (019-19741, Wako Chemicals, VA, USA); NeuN, a marker of mature neurons (Millipore, MA, USA); and β-actin (sc-81178, Santa Cruz, USA).

Cell lysates were separated using sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The antibodies used were as follows: anti-phospho-Y397-Fak (ab81298) and anti-FAK (ab131435) from Abcam (Cambridge, MA, USA); anti-Tyr416-phosphor-c-Src (2010s), anti-c-Src (2108s), anti-phosphoERK1/2 (T202/Y204), and anti-ERK1/2 (4370s) from Cell Signaling Technology (Danvers, MA, USA); anti-integrin α3 (21992-1-AP) from Proteintech (Rosemont, IL, USA); and β-actin (sc-81178) from Santa Cruz (Santa Cruz, CA, USA). Primary and secondary antibodies were incubated with the polyvinylidene difluoride membranes using standard techniques. Immunodetection was accomplished using enhanced chemiluminescence. Chemiluminescence was measured with a quantitative digital imaging system (Quantity One; BioRad, Hercules, CA, USA) allowing to check for saturation. Overall emitted photons were quantified for each band, particularly for loading controls, which were homogeneously loaded.

For Villin-Cre; Tsc1^f/f mice, villus tissues were scraped from the small intestines. For Lgr5-CreERT; Tsc1^f/f mice, crypts were isolated. In these experiments, the mice were starved overnight and harvested in the following morning. These samples were lysed in T-PER™ Tissue protein extraction reagent purchased from Thermo Scientific (78510) containing 1 mM PMSF and 1 μg/ml aprotonin, leupeptin, and pepstatin. Cells were lysed in TNEN buffer containing 1 mM PMSF and 1 μg/ml aprotonin, leupeptin, and pepstatin. The protein concentration was determined by a Bio-Rad assay. Proteins were resolved by SDS-PAGE and transferred to polyvinylidenedifluoride membranes (Millipore). Antibodies against MKK6 (9264, 1:1000), p-p38 (9211, 1:1000), p38 (9212, 1:1000), p-Erk1/2 (9106, 1:1000), Erk1/2 (9102, 1:1000), cyclin E (4129, 1:1000), p53 (2524, 1:1000), p-S6 (1211, 1:1000), p-p70S6K (9234, 1:1000), p-4E-BP1 (2855, 1:1000), 4E-BP1 (9644, 1:1000), p-mTOR (5536, 1:1000), and mTOR (2983, 1:1000) were purchased from Cell Signaling Technology. Antibodies against β-actin (sc81178, 1:1000), p16 (sc1207, 1:1000) and PCNA (sc-56, 1: 1000) were purchased from Santa Cruz Biotechnology. The protein bands were quantitated using the software provided by FluoChem M system (Protein Simple). Uncropped western blot images can be found in Supplementary Fig. 11.